Use of meganucelases for inducing homologous recombination ex vivo and in toto in vertebrate somatic tissues and application thereof

ABSTRACT

A single chain homing endonuclease, comprising a first variant of I-CreI having the amino acid sequence of accession number pdb 1g9y and a second variant of I-CreI variant having the amino acid sequence of accession number pdb 1g9y in a single polypeptide.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 13/649,093,filed Oct. 10, 2012, which is a continuation of U.S. Ser. No.10/543,557, which entered the U.S. National Stage on Jul. 27, 2005, as aNational Stage (371) application of PCT/IB04/00848 (compliance with 35U.S.C. §371(c) on Mar. 14, 2006), filed on Jan. 28, 2004, and claimspriority to U.S. 60/491,535, filed on Aug. 1, 2003, and U.S. 60/442,911,filed on Jan. 28, 2003.

FIELD OF THE INVENTION

The present invention relates to the use of meganucleases for inducinghomologous recombination ex vivo and in toto in vertebrate somatictissues and to its application for genome engineering and gene therapy.

BACKGROUND OF THE INVENTION

Homologous gene targeting has been widely used in the past to obtainsite-specific and precise genome surgery (Thomas and Capecchi, 1986,Nature, 324, 34-8; Thomas et al., 1986, Cell, 44, 419-28; Thomas andCapecchi, 1986, Cold Spring Harb Symp Quant Biol, 51 Pt 2, 1101-13;Doetschman et al.,1988, PNAS, 85, 8583-7). Homologous gene targetingrelies on the homologous recombination machinery, one of the endogenousmaintenance systems of the cell. Since this system has been wellconserved throughout evolution, gene targeting could be used inorganisms as different as bacteria, yeast, filamentous fungi, mammals,insects, and plants.

One direct application is the modulation of gene expression by modifyingthe regulatory sequences surrounding the gene (EP 419621; US patents:U.S. Pat. Nos. 6,528,313; 6,528,314; 5,272,071; 5,641,670). Correctionof mutated genes by homologous recombination is another application(Fischer et al., 2002, Isr Med Assoc J, 4, 51-4). A deleterious mutationcan often be complemented by the introduction of a wild type geneanywhere else in the genome. However, there are three major drawbacks insuch an approach (random transgenesis). First, the mutated gene is stillpresent. Certain mutation will result in a gain of function, that willnot be complemented by the wild type gene, or that can at leastinterfere with the wild type gene. Second, gene expression often dependson very long tracts of surrounding sequences. In higher eukaryotes,these sequences can span over several hundreds of kbs, and are necessaryfor the precise tuning of gene expression during the cell cycle,development, or in response to physiological signals. Even thoughtransgenic sequences involve most of the time a few kbs, there is no waythey can restore a fully wild type phenotype. This problem can howeverbe alleviated by transformation with very large sequences (BAC), but itrequires additional skills. Third, random transgenesis results ininsertions anywhere in the genome, with a non-nul probability of adeleterious effect: insertion in a gene will disrupt the gene or itsproper regulation. Such deleterious effect have been fully illustratedrecently in gene therapy trials for SCID patients (Fischer et al.,precited), which resulted in cases of leukemia-like syndromes, probablyas a consequence of deleterious insertions of the virus-bornetransgenes.

In contrast with random transgenesis, homologous recombination allowsthe precise modification of a chromosomal locus: it can result in genedeletion, gene insertion, or gene replacement, depending on thetargeting vector. In addition, subtle changes can be introduced in aspecific locus, including the modification of coding and regulatorysequences (EP 419621; US Patents: U.S. Pat. Nos. 6,528,313; 6,528,314;5,272,071; 5,641,670; 6,063,630).

These specific advantages should make homologous gene targeting auniversal tool for genome engineering, and the only safe methodology forgene therapy.

However, the use of homologous recombination is limited by its poorefficiency in most cells. Although homologous gene targeting isextremely efficient in the yeast Saccharomyces cerevisiae (Paques andHaber, 1999, Microbiol Mol Biel Rev, 63, 349-404), the mossPhyscomitrella patens (Schaefer and Zryd, 1997, Plant J, 11, 1195-206),certain mutant Escherichia coli strains (Murphy, 1998, J. Bacteriol,180, 2063-71; Zhang et al., 1998, Nat Genet, 20, 123-8), and in aviancell lines such as DT40 (Buerstedde and Takeda, 1991, Cell, 67, 179-88),its efficiency remains extremely low in most cells and organisms. Forexample in cultured mammalian cells, such recombination events usuallyoccur in only one in ten thousands cells which have taken up therelevant correcting or targeting DNA.

As a consequence, many approaches have been used to improve theefficiency of homologous gene targeting. Chimeraplasty (Yoon et al.,1996, PNAS, 93, 2071-6), Small Fragment Homologous Recombination (Gonczet al., 2002, Gene Ther, 9, 691-4) and Triplex Forming Oligonucleotides(Gorman and Glazer, 2001, Curr Mol Med, 1, 391-9) are as many examples.However, the most robust and efficient way to improve homologous genetargeting remains to deliver a DNA double-strand break (DSB) in thelocus of interest (US Patents: U.S. Pat. Nos. 5,474,896; 5,792,632;5,866,361; 5,948,678; 5,948,678, 5,962,327; 6,395,959; 6,238,924;5,830,729). This method improves the targeting efficiency by severalorders of magnitude in mammalian cells (Donoho et al., 1998, Mol CellBiol, 18, 4070-8; Rouet et al., 1994, Mol Cell Biol, 14, 8096-106;Choulika et al., 1995, Mol Cell Biol, 15, 1968-73; Cohen-Tannoudji etal., 1998, Mol Cell Biol, 18, 1444-8; Porteus and Baltimore, 2003,Science, 300, 763; Porteus et al., 2003, Mol Cell Biol, 23, 3558-65;Miller et al., 2003, Mol Cell Biol, 23, 3550-7) and allows genetargeting in plants (Puchta et al., 1993, Nucleic Acids Res, 21,5034-40) and Drosophila (Bibikova et al., 2003, Science, 300, 764).

Therefore, the introduction of the double-strand break is accompanied bythe introduction of a targeting segment of DNA homologous to the regionsurrounding the cleavage site, which results in the efficientintroduction of the targeting sequences into the locus (either to repaira genetic lesion or to alter the chromosomal DNA in some specific way).Alternatively, the induction of a double-strand break at a site ofinterest is employed to obtain correction of a genetic lesion via a geneconversion event in which the homologous chromosomal DNA sequences froman other copy of the gene donates sequences to the sequences where thedouble-strand break was induced. This latter strategy leads to thecorrection of genetic diseases either in which one copy of a defectivegene causes the disease phenotype (such as occurs in the case ofdominant mutations) or in which mutations occur in both alleles of thegene, but at different locations (as is the case of compoundheterozygous mutations), (WO 96/14408; WO 00/46386; US 5,830,729;Choulika et al., precited; Donoho et al., precited; Rouet et al.,precited).

However, the delivery of site-specific DSBs proved to be anotherchallenge. It requires the use of site-specific endonucleasesrecognizing large sequences. Such very rare-cutting endonucleasesrecognizing sequences larger than 12 base pairs are calledmeganucleases. Ideally, one would like to use endonucleases cutting onlyonce in the genome of interest, the cleavage being limited to the locusof interest.

In the wild, such endonucleases are essentially represented by homingendonucleases (Chevalier and Stoddard, 2001, N.A.R., 29, 3757-74).Homing endonucleases are found in fungi, algae, eubacteria and archae,and are often encoded in mobile genetic elements. Their cleavageactivities initiate the spreading of these mobile elements by homologousrecombination. The biology of HO (Haber, 1998, Annu Rev Genet, 32,561-99; Haber, 1995, Bioessays, 17, 609-20), I-SceI (Jacquier and Dujon,1985, Cell, 41, 383-94; Fairhead and Dujon, 1993, Mol Gen Genet, 240,170-8; Colleaux et al., 1988, PNAS, 85, 6022-6; Perrin et al., 1993,Embo J, 12, 2939-47; Plessis et al., 1992, Genetics, 130, 451-60) andI-TevI endonucleases (Bell-Pedersen et al., 1989, Gene, 82, 119-26;Bell-Pedersen et al., 1990, Nucleic Acids Res, 18, 3763-70; Mueller etat., 1996, Genes Dev, 10, 2158-66) are among the many paradigms for suchDSB-induced recombination events.

HO and I-SceI have been used to induce homologous gene targeting inyeast (Haber, 1995, precited; Fairhead and Dujon, 1993, precited;Plessis et al., 1992, precited; U.S. Pat. Nos. 5,792,632 and 6,238,924),in cultured mammalian cells (Donoho et al.; Rouet et al.; Choulika etal.; Cohen-Tannoudji et al., precited; Patents: U.S. Pat. Nos.5,792,632; 5,830,729 and 6,238,924) and plants (Puchta et al., 1996,PNAS, 93, 5055-60; U.S. Pat. Nos. 5,792,632 and 6,238,924).Meganucleases have also been used to trigger various intra- andinterchromosomal rearrangements based on DSB-induced homologousrecombinations in bacteria (Posfai et al., 1999, N.A.R., 27, 4409-15),yeast (Paques and Haber, 1999, Microbiol Mol Biol Rev, 63, 349-404),plants (Siebert and Puchta, 2002, Plant Cell, 14, 1121-31; Chiurazzi etal., 1996, Plant Cell, 8, 2057-66; Puchta, 1999, Genetics, 152,1173-81), insects (Rong et al., 2002, Genes Dev, 16, 1568-81) andcultured mammalian cells (Lin and Waldman, 2001, Genetics, 158,1665-74;Liang et al., 1998, PNAS, 95, 5172-7).

Group II introns proteins can also be used as meganucleases. The biologyof these proteins is much more complex than the biology of homingendonucleases encoded by group I introns and inteins (Chevalier andStoddard, precited). The protein is involved in intron splicing, andforms a ribonucleic particle with the spliced RNA molecule. This complexdisplays different activities including reverse splicing (of the RNAintron in a DNA strand from the target gene), nicking (of the second DNAstrand in the novel gene) and reverse transcriptase (which copies theinserted RNA into a DNA strand). The final insertion of the intron intothe target gene depends on all these activities. These proteins seem toinduce homologous recombination, with a DSB intermediate, when thereverse transcriptase activity is mutated (Karberg et al., 2001, Nat.Biotechnol, 19, 1162-7).

Unfortunately, this method of genome engineering by using naturalmeganucleases for inducing homologous recombination by a double-strandbreak is limited by the introduction of a recognition and cleavage siteof said natural meganuclease at the position where the recombinationalevent is desired.

Up today, in a first approach for generating new megnucleases(artificial or man-made meganucleases), some chimeric restrictionenzymes have been prepared through hybrids between a DNA-binding domain(namely a zinc finger domain) and a catalytic domain (the non-specificDNA-cleavage domain from the natural restriction enzyme Fok I), (Smithet al, 2000, N.A.R, 28, 3361-9; Smith et al., 1999, Nucleic Acids Res.,27, 274-281; Kim et al., 1996, PNAS, 93, 1156-60; Kim & Chandrasegaran,1994, PNAS, 91, 883-7; WO 95/09233; WO 94/18313; U.S. Pat. No.5,436,150). The resulting so-called Zinc-finger nucleases have been usedto induce tandem repeat recombination in Xenopus oocytes (Bibikova etal., 2001, Mol Cell Biol, 21, 289-97), and homologous gene targeting incultured mammalian cell lines (Porteus and Baltimore, precited) andDrosophila (Bibikova et al., precited).

Another approach consisted of embedding DNA binding and catalyticactivities within a single structural unit, such as a type IIrestriction endonuclease. However, efforts to increase the length ofrecognition sequence or alter the specificity of these enzymes haveresulted in the loss of catalytic activity or overall diminution ofspecificity due to the tight interdependence of enzyme structure,substrate recognition and catalysis (Lanio et al., 2000, Protein Eng.,13, 275-281).

Based on homing endonuclease, Chevalier et al. (2002, Molecular Cell,10, 895-905) have also generated an artificial highly specificendonuclease by fusing domains of homing endonucleases I-Dmo I and I-CreI. The resulting enzyme binds a long chimeric DNA target site andcleaves it precisely at a rate equivalent to its natural parents.However, this experiment leads to one endonuclease with a newspecificity but it is not applicable to find an endonuclease thatrecognizes and cleaves any desired polynucleotide sequence.

Fusions between nucleic acids and chemical compounds are another classof artificial meganucleases, wherein DNA binding and specificity rely onan oligonucleotide and cleavage on a chemical compound tethered to theoligonucleotide. The chemical compounds can have an endogenous cleavageactivity, or cleave when complexed with topoisomerases (Arimondo et al.,2001, Angew Chem Int Ed Engl, 40, 3045-3048; Arimondo and Helene, 2001,Curr Med Chem Anti-Cane Agents, 1, 219-35).

Thus, meganuclease-induced recombination appears to be an extremelypowerful tool for introducing targeted modifications in genomes. Inaddition, the development of new meganucleases able to cleave DNA at theposition where the recombinational event is desired, for example derivedfrom Zinc-finger nucleases, or from natural homing endonucleases, wouldallow targeting at any given locus at will and with a reasonableefficiency.

Nevertheless, it clearly emerges from the above analysis of the priorart that the use of this technology in animals has so far been mostlylimited to its applications in vitro or ex vivo in cultured cells,except in the case of Drosophila (Bibikova et al. 2003, precited), whereit could be used to induce recombination in a living animal, in thegermline and somatic tissues.

It would be extremely advantageous to be able to use this technology toinduce recombination in a whole organism, in the somatic tissues:

-   -   This could be used for tissue-specific genome engineering in        animal models or foreign sequences excision in        genetically-modified organisms (once the trait depending on        these foreign sequences is not useful anymore). DSBs between two        tandem repeats induce very high levels of homologous        recombination resulting in deletion of one repeat together with        all the intervening sequences (Paques and Haber, 1999, Microbiol        Mol Biol Rev, 63, 349-404), and this can easily be used for the        removal of any transgene with an appropriate design.    -   One other major application would be the use of        meganuclease-induced recombination in gene therapy. In a number        of cases, an ex vivo approach could be used: precursor stem        cells would be taken from the patients, healed ex vivo, and        grafted back in the deficient tissue. So far, ex vivo techniques        have been mostly used with blood cells in SCID and other        syndromes (although random insertion was used instead of        homologous recombination (Fischer et al., precited). The        manipulation of stem cells makes it an attractive approach for        other tissues. However, the use of meganuclease-induced        recombination in toto would bypass the ex vivo steps and enlarge        the range of tissues that can be treated.

There are however two major reasons why this approach is notstraightforward:

-   -   First, this would require the delivery of a meganuclease in the        appropriate tissue.    -   Second, cells in a living organism do not necessarily behave as        cultured cells or germinal cells. Cultured cells and early (and        sometimes late) germ cells are dividing cells, going through G1,        S, G2, and M phases. In contrast, most cells in an adult animal        are differentiated cells, stuck in a GO phase. Many results        indicate and/or suggest that homologous recombination does not        have the same efficiency in all phases of the cell cycle (Takata        et al., 1998, Embo J, 17, 5497-508; Kadyk and Hartwell, 1992,        Genetics, 132, 387-402; Gasior et al., 2001, PNAS, 98, 8411-8;        Essers et al.,1997,Cell, 89,195-204). In general, the different        tissues might have distinct proficiencies for homologous gene        conversions. Therefore, it is not clear whether gene targeting        and meganuclease-induced genome engineering by homologous        recombination could be used in whole organisms, or even for ex        vivo approaches, which relies on specific cell types for which        recombination proficiencies are largely unknown.

Surprisingly, by using appropriate targeting constructs and meganucleaseexpression vectors, the Inventors have shown that meganucleases areindeed able to induce targeted homologous recombination ex vivo and intoto, in vertebrate somatic tissues.

Accordingly, meganucleases can be used for repairing a specificsequence, modifying a specific sequence, for attenuating or activatingan endogenous gene of interest, for inactivating or deleting anendogenous gene of interest or part thereof, for introducing a mutationinto a site of interest or for introducing an exogenous gene or partthereof, in vertebrate somatic tissues.

Therefore, these results establish a basis for efficient site-specificgenomic manipulation in mammalian somatic tissues for experimentalpurposes and raise the possibility of therapeutically correctingmutations by gene targeting.

DETAILED DISCUSSION OF THE INVENTION

Thus, the purpose of the present invention is to use meganucleases forinducing homologous recombination ex vivo and in toto in vertebratesomatic tissues.

Applications are in different fields: research, including animal modelsgeneration (tissue specific genome surgery: knock-in or knock-out);agricultural biotechnology (addition or removal of a trait, markerexcision, protein production) and therapeutics (gene therapy: generepair ex vivo and in toto and antiviral therapy: excision of virus exvivo and in toto).

Accordingly, the present invention relates to the use of at least onemeganuclease for the preparation of a medicament for preventing,improving or curing a genetic disease in a vertebrate in need thereof;said medicament being administered by any means to said vertebrate.

The invention, also concerns the use of at least one meganuclease forthe preparation of a medicament for preventing, improving or curing adisease caused by an infectious agent that presents a DNA intermediate,in a vertebrate in need thereof; said medicament being administered byany means to said vertebrate. Preferably, said infectious agent is avirus.

Another object of the present invention is the use of at least onemeganuclease for genome engineering of non-human vertebrate somatictissues, for non-therapeutic purpose, by introducing said meganucleaseinto the body of said non-human vertebrate.

Definitions

In the present application, by “meganuclease” is intended adouble-stranded endonuclease having a large polynucleotide recognitionsite, at least 12 bp, preferably from 12 by to 60 bp. Said meganucleaseis also called rare-cutting or very rare-cutting endonuclease. Saidmeganuclease is either monomeric or dimeric. It includes any naturalmeganuclease such as a homing endonuclease, but also any artificial orman-made meganuclease endowed with such high specificity, either derivedfrom homing endonucleases of group I introns and inteins, or otherproteins such as Zinc-Finger proteins or group II intron proteins, orcompounds such as nucleic acid fused with chemical compounds.

In particular, artificial meganucleases include the so-called“custom-made meganuclease” which is a meganuclease derived from anyinitial meganuclease, either natural or not, presenting a recognitionand cleavage site different from the site of the initial one;zinc-finger nucleases may also be considered as custom-mademeganucleases. By “different” is intended that the custom-mademeganuclease cleaves the novel site with an efficacy at least 10 foldmore than the natural meganuclease, preferably at least 50 fold, morepreferably at least 100 fold. “Natural” refers to the fact that anobject can be found in nature. For example, a meganuclease that ispresent in an organism, that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnatural.

By “in toto” is intended that the homologous recombination event inducedby the meganuclease takes place in vivo in the body of a vertebrate;said meganuclease is introduced into the body of said vertebrate by anyconvenient mean.

By “ex vivo” is intended that the homologous recombination event inducedby the meganuclease takes place in somatic cells removed from the bodyof a vertebrate; said meganuclease is introduced (ex vivo) into thecells of said vertebrate by any convenient mean and the modified cellsare then returned into the body of said vetebrate.

By “somatic tissue” is intended any tissue within the body of anorganism including any type of cells from the precursor cells (stemcells) to the fully differentiated cells, with the exception of the germline cells.

“Identity” refers to sequence identity between two nucleic acidmolecules or polypeptides. Identity can be determined by comparing aposition in each sequence which may be aligned for purposes ofcomparison. When a position in the compared sequence is occupied by thesame base, then the molecules are identical at that position. A degreeof similarity or identity between nucleic acid or amino acid sequencesis a function of the number of identical or matching nucleotides atpositions shared by the nucleic acid sequences. Various alignmentalgorithms and/or programs may be used to calculate the identity betweentwo sequences, including FASTA, or BLAST which are available as a partof the GCG sequence analysis package (University of Wisconsin, Madison,Wisc.), and can be used with, e.g., default settings.

By “ homologous ” is intended a sequence with enough identity to anotherone to lead to a homologous recombination between sequences, moreparticularly having at least 95% identity, preferably 97%, and morepreferably 99%.

The phrases “site of interest”, “target site” and “specific site”, asused herein, refer to a distinct DNA location, preferably a chromosomallocation, at which a double stranded break (cleavage) is to be inducedby the meganuclease.

As used herein, the term “individual” includes mammals, as well as othervertebrates (e.g., birds, fish and reptiles). The terms “mammal” and“mammalian”, as used herein, refer to any vertebrate animal, includingmonotremes, marsupials and placental, that suckle their young and eithergive birth to living young (eutharian or placental mammals) or areegg-laying (metatharian or nonplacental mammals). Examples of mammalianspecies include humans and other primates (e.g., monkeys, chimpanzees),rodents (e.g., rats, mice, guinea pigs) and ruminants (e.g., cows, pigs,horses).

By “genetic disease” is intended any disease, partially or completely,directly or indirectly, due to an abnormality in one or several genes.Said abnormality can be a mutation, an insertion or a deletion. Saidmutation can be a punctual mutation. Said abnormality can affect thecoding sequence of the gene or its regulatory sequence. Said abnormalitycan affect the structure of the genomic sequence or the structure orstability of the encoded mRNA. Said genetic disease can be recessive ordominant.

The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked.

Meganucleases

In another embodiment of the above uses according to the invention, saidmeganuclease is selected from the group consisting of a homingendonuclease, a zinc-finger nuclease or a meganuclease variant.

-   -   homing endonuclease are as described in Chevalier and Stoddard,        precited.

Custom-made Meganucleases

Zinc-Finger Nuclease

Meganuclease based on Zinc-Finger domains have the structure describedby Smith et al., precited. The meganuclease is a heterodimer of twofusion protein. Each fusion protein includes a DNA-binding domainderived from Zif268 (or other zinc-finger proteins), tethered to anuclease domain (derived from the FokI endonuclease or otherendonucleases) through a linker. The DNA target site includes twoexternal regions of 9 bp, bound by the DNA binding domains, and acentral spacer region of 0-15 bp. In each monomer, the DNA bindingZinc-Finger domain has been selected to bind one of the 9 by externalregions, as described by Isalan and Choo (2001, Methods Mol Biol,148,417-29), Isalan et al. (2001, Nat. Biotechnol, 19, 656-60) andIsalan and Choo (2001, Methods Enzymol, 340,593-609). Selection can bemade by phage display, as described by the authors, but other methodssuch as screening in yeast or with a bacterial two-hybrid system canalso be used, as described by Young et al. (2000, PNAS, 97, 7382-7) andBae et al. (2003, Nat Biotechnol, 21,275-80). Also, to enhancespecificity, DNA binding domains encompassing 6 Zinc Finger motifs canbe used, as described by Klug and collaborators (Moore et al., 2001,

PNAS, 98,1432-6; Papworth et al., 2003, PNAS, 100,1621-6; Reynolds etal., 2003, PNAS, 100,1615-20; Moore et al., 2001, PNAS, 98, 1437-41).However, if the endonucleolytic activity relies on a Fokl domain, twosuch monomers have to be used, each one bound to a Fokl catalytic site:each Fokl catalytic domain cleaving only one strand, it takes two suchdomains to obtain a double-strand cleavage.

Meganuclease Variants

Custom-made meganuclease is defined as a meganuclease able to cleave atargeted DNA sequence. This definition includes any meganuclease variantproduced by a method comprising the steps of preparing a library ofmeganuclease variants and isolating, by selection and/or screening, thevariants able to cleave the targeted DNA sequence. Said custom-mademeganuclease which is derived from any initial meganuclease byintroduction of diversity, presents a recognition and cleavage sitedifferent from the site of the initial one.

The diversity could be introduced in the meganuclease by any methodavailable for the man skilled in the art. Preferably, the diversity isintroduced by targeted mutagenesis (i.e. cassette mutagenesis,oligonucleotide directed codon mutagenesis, targeted randommutagenesis), by random mutagenesis (i.e. mutator strains, Neurosporacrassa system (U.S. Pat. No. 6,232,112; WO01/70946, error-prone PCR), byDNA shuffling, by directed mutation or a combination of thesetechnologies (See Current Protocols in Molecular Biology, Chapter 8“Mutagenesis in cloned DNA”, Eds Ausubel et al., John Wiley and Sons).The meganuclease variants are preferably prepared by the targetedmutagenesis of the initial meganuclease. The diversity is introduced atpositions of the residues contacting the DNA target or interacting(directly or indirectly) with the DNA target. The diversity ispreferably introduced in regions interacting with the DNA target, andmore preferably introduced at the positions of the interacting aminoacids. In libraries generated by targeted mutagenesis, the 20 aminoacids can be introduced at the chosen variable positions. Preferably,the amino acids present at the variable positions are the amino acidswell-known to be generally involved in protein-DNA interaction. Moreparticularly, these amino acids are generally the hydrophilic aminoacids. More preferably, the amino acids present at the variablepositions comprise D, E, H, K, N, Q, R, S, T, Y. Optionally, the aminoacids present at the variable positions are selected from the groupconsisting of D, E, H, K, N, Q, R, S, T, Y. Synthetic or modified aminoacids may also be used.

One preferred way to generate a directed library is the use ofdegenerated codons at the positions where diversity has to beintroduced. Several types of degenerated codons could be used. Adegenerated codon N N K ([ATCG] [ATCG] [TG]) leads to 32 differentcodons encoding the 20 amino acids and one stop. A degenerated codon N VK ([ATCG] [ACG] [TG]) leads to 24 different codons encoding the 15 aminoacids and one stop. A degenerated codon V V K ([ACG] [ACG] [TG]) leadsto 18 different codons encoding the 12 amino acids (A, D, E, G, H, K, N,P, Q, R, S, T) and no stop. A degenerated codon R V K ([AG] [ACG] [TG])leads to 12 different codons encoding the 9 amino acids (A, D, E, G, K,N, R, S, T).

Preferably, a degenerated codon V V K ([ACG] [ACG] [TG]) leading to 18different codons encoding the 12 amino acids (A, D, E, G, H, K, N, P, Q,R, S, T) is used for generating the library. Indeed, the V V Kdegenerated codon does not contain any stop codon and comprises all thehydrophilic amino acids.

If a directed library is generated, knowledge on amino acids interactingwith the DNA target is useful. This knowledge could be provided, forexample, by X-ray cristallography, Alanine scanning, or cross-linkingexperiments. The amino acids interacting with the DNA target can also bededuced by sequence alignment with a homologous protein.

The custom-made meganuclease is derived from any initial meganuclease.

Optionally, the initial meganuclease is selected so as its naturalrecognition and cleavage site is the closest to the targeted DNA site.Preferably, the initial meganuclease is a homing endonuclease, asspecified, in the here above definitions. Homing endonucleases fall into4 separated families on the basis of well conserved amino acids motifs,namely the LAGLIDADG family, the GIY-YIG family, the His-Cys box family,and the HNH family (Chevalier et al., 2001, N.A.R, 29, 3757-3774).

The detailed three-dimensional structures of several homingendonucleases are known, namely I-Dmo I, PI-Sce I, PI-Pfu I, I-Cre I,I-Ppo I, and a hybrid homing endonuclease I-Dmo I/I-Cre I called E-Dre I(Chevalier et al., 2001, Nat Struct Biol, 8, 312-316; Dunn et al., 1997,Cell, 89, 555-564; Heath et al., 1997, Nat Struct Biol, 4, 468-476; Huet al., 2000, J Biol Chem, 275, 2705-2712; Ichiyanagi et al., 2000, JMol Biol, 300, 889-901; Jurica et al., 1998, Mol Cell, 2, 469-476;Poland et al., 2000, J Biol Chem, 275, 16408-16413; Silva et al., 1999,J Mol Biol, 286, 1123-1136; Chevalier et al., 2002, Molecular Cell, 10,895-905).

The LAGLIDADG family is the largest family of proteins clustered bytheir most general conserved sequence motif: one or two copies of atwelve-residue sequence: the di-dodecapeptide, also called LAGLIDADGmotif. Homing endonucleases with one dodecapeptide (D) are around 20 kDain molecular mass and act as homodimer. Those with two copies (DD) rangefrom 25 kDa (230 AA) to 50 kDa (HO, 545 AA) with 70 to 150 residuesbetween each motif and act as monomer. Cleavage is inside therecognition site, leaving 4 nt staggered cut with 3′OH overhangs. I-CeuI, and I-Cre I illustrate the homodimeric homing endonucleases with oneDodecapeptide motif (mono-dodecapeptide). I-Dmo I, I-Sec I, PI-Pfu I andPI-Sce I illustrate monomeric homing endonucleases with twoDodecapeptide motifs.

The initial LAGLIDADG homing endonuclease can be selected from the groupconsisting of: I-Sce I, I-Chu I, I-Dmo I, I-Cre I, I-Csm I, PI-Sce I,PI-Tli I, PI-Mtu I, I-Ceu I, I-Sce II, I-Sce III, HO, PI-Civ I, PI-CtrI, PI-Aae I, PI-Bsu I, PI-Dha I, PI-Dra I, PI-May I, PI-Mch I, PI-Mfu I,PI-Mfl I, PI-Mga I, PI-Mgo I, PI-Min I, PIMka I, PI-Mle I, PI-Mma I,PI-Msh I, PI-Msm I, PI-Mth I, PI-Mtu I, PI-Mxe I, PI-Npu I, PI-Pfu I,PI-Rma I, PI-Spb I, PI-Ssp I, PI-Fac I, PI-Mja I, PI-Pho I, PI-Tag I,PI-Thy I, PI-Tko I, and PI-Tsp I; preferably, I-Sce I, I-Chu I, I-Dmo I,I-Cre I, I-Csm I, PI-Sce I, PI-Pfu I, PI-Tli I, PI-Mtu I, and I-Ceu I;more preferably, I-Dmo I, I-Cre I, PI-Sce I, and PI-Pfu I; still morepreferably I-Cre I.

The four structures of LAGLIDADG homing endonucleases, namely those ofI-Dmo I, PI-Sce I, PI-Pfu I, and I-Cre I, reveal the functionalsignificance of the LAGIDADG motif, and the nature of the DNA-bindinginterface. The core αββαββα fold of the homodimer homing endonuclease isrepeated twice in the monomer homing endonuclease and confers upon themonomer a pseudo-dimeric structure. The first α-helix of each domain orsubunit contains the defining LAGLIDADG motif The two LAGLIDADG helicesof each protein form a tightly packed dimer or domain interface. The DNAbinding interface is formed by the four (β-strands of each domain orsubunit that fold into an antiparallel β-sheet. A minimal DNA bindingmoiety could be defined in the LAGLIDADG homing endonucleases as a(β-hairpin (2β-strands connected by a loop or turn), two such β-hairpinsbeing connected into the 4-stranded (β-sheet.

Each domain or subunit interacts with a half recognition site. The<<external>> quarter recognition site can be defined by its interactionwith only one of the 2 β-hairpins of each domain or subunit.

Therefore, meganuclease variants derived from LAGLIDADG homingendonuclease can be fragmented in several directed libraries. Thisfragmented approach for the evolution of an initial meganuclease allowsthe introduction of a greater diversity (more amino acids at a positionand/or more diversificated positions). In each library, the diversity isintroduced only in the region involved in the inter-action with a halfor a quarter recognition site, the targeted DNA being modified only forthe part interacting with the region comprising the introduceddiversity. More particularly, if a new half site is searched for, thenthe diversity is preferably introduced in the 4-stranded (β-sheet of onedomain or subunit, more preferably at the positions of the DNAinteracting amino acids in this structure. If a new quarter site issearched for, then the diversity is introduced in the correspondingβ-hairpin, more preferably at the positions of the DNA interacting aminoacids of this structure.

Preferably, a set of libraries covers the entire targeted DNA site.Hence, if the libraries comprise diversity only in the regioninteracting with a half-site, at least two libraries, preferably two,are necessary. However, if the initial meganuclease is a dimer, onelibrary is enough with a half-site approach. If the libraries comprisediversity only in the region interacting with a quarter site, at leastfour libraries, preferably four, are necessary. If the initialmeganuclease is a dimer, two libraries can be enough with a quarter siteapproach.

After the selection or screening of the primary libraries, the selectedelements from the primary libraries are fused or combined in asubsequent library for a new cycle of selection. For example, twolibraries can be fused by shuffling. A new cycle of selection could bethen done on the whole targeted DNA site. Optionally, the new cycle ofselection can be done on a half targeted DNA site if the first librariesare based on a quarter site. Subsequently, the results of the selectionand/or screening of the half site are combined to give a final librarywhich can be screened for the whole targeted DNA site.

Alternatively, the best elements from each libraries are joined togetherin order to obtain a meganuclease able to bind and cleave the targetedDNA site,

In another approach, a library with diversity located only in the regioninvolved in the interaction with a half or a quarter recognition site isprepared. Then, after selection or screening of this library, theselected elements from the library are modified such as to introducediversity in another region involved in the interaction with recognitionsite, leading to a subsequent library. Libraries are generated until thecomplete targeted DNA site is bound and cleaved by the selectedmeganuclease.

More specifically, for the dimeric homing endonuclease (such as I-Cre Iand I-Ceu I), a library can be generated by introducing diversity onlyin the region interacting with a half-site, a half site corresponding toone monomer of the initial homing endonuclease. This library can be usedfor selection and/or screening on each half sites of the target DNAsequence. When positive elements from the library have been selected foreach half sites, a variant for the first half site and a variant for theother half site are brought together for binding and cleaving the wholetarget DNA sequence. Alternatively, the positive variants can beintroduced in a single chain meganuclease structure. As described inExample 1, a single chain meganuclease is an enzyme in which the twomonomers of the initial dimeric homing endonuclease are covalently boundby a linker.

If an approach by a quarter site is chosen from an initial dimer homingendonuclease, at least two libraries are generated by introducingdiversity only in the region involved in the interaction with eachquarter recognition sites. After the selection or screening of theprimary libraries, the selected variants from the primary libraries arefused in a subsequent library for a new cycle of selection on the halfsite. Alternatively, the best elements from each libraries are joinedtogether to obtain a monomer able to bind the half site. Otherwise, alibrary with diversity only in the region involved in the interactionwith a quarter recognition site is prepared. Then, after selection orscreening of this library, the selected elements from the library aremodified such as to introduce diversity in the region involved in theinteraction with the other quarter site, leading to a subsequentlibrary. The selection and/or screening of this second library lead tothe variants monomer able to bind the half site. When positive elementsfrom the library have been selected for each half sites, a variant forthe first half site and a variant for the other half site are broughttogether for binding and cleaving the target DNA sequence.Alternatively, the positive variants can be introduced in a single chainmeganuclease structure.

Preferably, the custom-made meganuclease which recognizes and cleaves adesired polynucleotide target is derived from the directed evolution ofthe homing endonuclease I-Cre I. As the homing endonuclease is ahomodimer, the approach in this case is based either on the halfrecognition site or on the quarter site.

The directed evolution is based on a library of I-Cre I variants. TheseI-Cre I variants present a diversity of amino acids at several positionspredicted to interact with the polynucleotide target.

The X-ray structure of I-Cre endonuclease with its DNA target predictedthat the following positions are involved: Q26, K28, N30, S32, Y33, Q38,Q44, R68, R70 and T140. Seligman et al (supra) showed that the positionsS32 and T140 appear to be relatively unimportant for DNA recognition.

A set of I-Cre I variants is prepared by introducing amino aciddiversity in positions selected from the group consisting of: Q26, K28,N30, S32, Y33, Q38, Q44, R68, R70 and T140. In a preferred embodiment, aset of I-Cre I variants is prepared by introducing diversity inpositions: a) Q26, K28, N30, Y33, Q38, Q44, R68, R70, T140; b) Q26, K28,N30, Y33, Q38, Q44, R68, R70; c) Q26, K28, N30, Y33, Q44, R68, R70; ord) Q26, K28, Y33, Q38, Q44, R68, R70. Preferably, a set of I-Cre Ivariants is prepared by introducing diversity in positions Q26, K28,N30, Y33, Q38, Q44, R68, and R70.

Optionally, the residue D75 of I-Cre I could be mutated in an unchargedamino acid such as N. Indeed, this amino acid has an interaction with 2residues which are preferably modified in the library. As this charge ispresent in the core of the structure, it could be preferable to abolishthis charge.

If the evolution approach of the homing endonuclease I-Cre I is based onthe quarter recognition site, replacing the DNA binding residuespresented by a β-hairpin (within the 4-stranded b-sheet) is a practicalsolution. As those residues are part of an element with limited length(i.e. less than 25 residue), they can be mutated together at once, forexample by cassette replacement. Visual inspection of structure lg9y,SEQ ID NO: 23, (I-CreI with its target double-stranded DNA) indicatesthat the first β-hairpin is a unique or major contributor to therecognition of the last six bases of the target (i.e. either bases −12to −7 or bases +7 to +12). Thus replacing the sequence from residue S22to residue Q44, more preferably from residue 124 to residue T42, shouldbe sufficient to specify new interaction specificity for the last sixbases of the target site. More preferably, the residues interactingdirectly with DNA should be modified: 124, Q26, K28, N30, S32, Y33, Q38,S40 and T42. Alternatively (or in addition), the turn at the middle ofthe β-hairpin, which interacts with the very end of the 24bp-long DNAtarget, may be replaced by a short and flexible loop that would betolerant to DNA bases substitution. For example, residues 30 to 36 couldbe replaced by 2, 3, 4, 5 or 6 glycine residues. This strategy is worthtesting with all meganucleases presenting a comparable 3D structure. Thesecond hairpin could be replaced similarly as a single unit (fromresidue Y66 to 177). However, while this hairpin interacts predominantlywith the internal quarter site (bases −6 to −1 or +1 to +6), otherresidues (i.e. S22, Q44 and T46) separated from the hairpin may play arole in directing the specificity of interaction. Thus, a library couldbe created by replacing residues Y66, R68, R70, V73, D75 and 177. Inparallel, S22, Q44 and T46 may either be left untouched, replaced bysmall polar amino acids (G, S or T; more preferably S or T), orrandomized to contribute to the library. Mutants selected from separatelibrary (the first wherein randomized residues are 124, Q26, K28, N30,S32, Y33, Q38, S40 and T42 and the second wherein randomized residuesare Y66, R68, R70, V73, D75 and 177) can be combined together bystandard DNA shuffling methods based on recombination at homologous DNAregions (i.e. the DNA coding for the region between residue 43 andresidue 65 is strictly conserved). However, if the second libraryincludes mutations of residues S22, Q44 and T46, recombination becomesimpractical, and more classical DNA/protein engineering is required.

If the evolution approach of the homing endonuclease I-Cre I is based onthe quarter recognition site, a library of I-Cre I variants is preparedby introducing diversity in positions selected from the group consistingof: a) 124, Q26, K28, N30, S32, Y33, Q38, S40 and T42; or b) Y66, R68,R70, V73, D75, and 177. In the alternative b), the diversity could bealso introduced in positions selected from the group consisting of: S22,Q44, and T46.

Alternatively, a custom-made meganuclease which recognizes and cleaves adesired polynucleotide target could be prepared by the directedevolution of single chain I-Cre I endonuclease. A set of single-chainI-Cre I variants is prepared by introducing amino acid diversity inpositions selected from the group consisting of:

Q26, K28, N30, S32, Y33, Q38, Q44, R68, R70, Q123, KI25, N127, S129,Y130, Q135, Q141, R165, R167.

Two properties of the meganuclease can be used for the steps ofselection and/or screening, namely the capacity to bind the targeted DNAsequence and the ability to cleave it.

The meganuclease variants can be selected and screened, or onlyscreened. The selection and/or screening can be done directly for theability of the meganuclease to cleave the targeted DNA sequence.Alternatively, the selection and/or screening can be done for thebinding capacity on the targeted DNA sequence, and then for ability ofthe meganuclease to cleave it. Preferably, the method to prepare acustom-made meganuclease comprises or consists of the following steps:

a) a selection step for the binding ability, a screening step for thebinding ability, a selection for the cleavage activity, and a screeningstep for the cleavage activity;

b) a selection step for the binding ability, a screening step for thebinding ability, and a screening step for the cleavage activity;

c) a selection step for the binding ability, a selection for thecleavage activity, and a screening step for the cleavage activity;

d) a screening step for the binding ability and a screening step for thecleavage activity;

e) a selection step for and a screening step for the cleavage activity;or,

f) a screening step for the cleavage activity.

More preferably, the method to prepare a custom-made meganucleasecomprises or consists of the following steps: a selection step for thebinding ability, a selection for the cleavage activity, and a screeningstep for the cleavage activity. A screening assay for the bindingability after a selection step based on the binding capacity can be donein order to estimate the enrichment of the library for meganucleasevariants presenting a binding capacity.

The selection and screening assays are performed on the DNA region inwhich a double stranded cleavage has to be introduced or a fragmentthereof

Preferably, the targeted sequences comprise at least 15 nucleotides,preferably 18 to 40, more preferably 18 to 30 nucleotides. In case ofdimeric meganuclease, the targeted DNA polynucleotide can be reduced toat least 8 nucleotides for binding only. Preferably, the targeted DNApolynucleotide length is less than 10 kb, preferably less than 3 kb,more preferably less than 1 kb. For the DNA binding assay, the targetedDNA polynucleotide length is preferably less than 500 bp, morepreferably less than 200 bp.

Any targeted sequence can be used to generate a custom-made meganucleaseable to cleave it according. Optionally, the targeted sequence is chosensuch as to present the most identity with the original recognition andcleavage site of the initial meganuclease. Therefore, the DNA region inwhich a double stranded break has to be introduced is analyzed to chooseat least 1, 2, 3 or 5 sequences of at least 15 nucleotides length,preferably 18 to 40, more preferably 18 to 30 nucleotides, having atleast 25% identity, preferably 50% identity and more preferably 75%identity with the original recognition and cleavage site of the initialmeganuclease.

The targeted DNA sequence is adapted to the type of meganucleasevariants library. If the library is based on a half site approach, thetargeted DNA sequence used for the selection/screening comprises onehalf original site and one half site of the desired DNA sequence. If thelibrary is based on a quarter site approach, the targeted DNA sequenceused for the selection/screening comprises three quarters of theoriginal site and one quarter site of the desired DNA sequence.

The meganuclease variants resulting from the selection and/or screeningsteps could optionally be an input for another cycle of diversityintroduction.

The positive meganuclease variants selected by the selection and/orscreening steps are validated by in vitro and/or ex vivo cleavage assay.

The selection and screening of meganuclease variants based on thebinding capacity has to be made in conditions that are not compatiblewith the cleavage activity. For example, most of homing endonucleasesneed manganese or magnesium for their cleavage activity. Therefore, thebinding assays on this type of homing endonuclease variants are donewithout manganese or magnesium, preferably replaced by calcium.

The binding selection assay is based on the enrichment of themeganuclease variants able to bind the targeted DNA polynucleotide.Therefore, the meganuclease variants encoded by the library areincubated with an immobilized targeted DNA polynucleotide so thatmeganuclease variants that bind to the immobilized targeted DNApolynucleotide can be differentially partitioned from those that do notpresent any binding capacity. The meganuclease variants which are boundto the immobilized targeted DNA polynucleotide are then recovered andamplified for a subsequent round of affinity enrichment andamplification. After several rounds of affinity enrichment andamplification, the library members that are thus selected can beisolated. Optionally, the nucleotide sequences encoding the selectedmeganuclease variants are determined, thereby identifying of themeganuclease variants able to bind the targeted DNA sequence.

The selection of meganuclease variants requires a system linkinggenotype and phenotype such as phage display (W091/17271, WO91/18980,and WO91/19818 and WO93/08278; the disclosures of which are incorporatedherein by reference), ribosome display (Hanes & Plückthun, PNAS, 1997,vol. 94, 4937-4942; He & Taussig, Nucl. Acids Res. (1997) vol. 25, p5132-5143) and mRNA-protein fusion (WO00/47775; U.S. Pat. No. 5,843,701;Tabuchi et al FEBS Letters 508 (2001) 309-312; the disclosures of whichare incorporated herein by reference).

Phage display involves the presentation of a meganuclease variant on thesurface of a filamentous bacteriophage, typically as a fusion with abacteriophage coat protein. The library of meganuclease variants isintroduced into a phage chromosome or phagemid so as to obtain a proteinfusion with a bacteriophage coat protein, preferably with the pIIIprotein. If the initial meganuclease is a homodimer, the monomervariants of the meganuclease are introduced so as to be displayed andthe constant monomer can be introduced so as to be produced in theperiplasm. The bacteriophage library can be incubated with animmobilized targeted DNA sequence so that elements able to bind the DNAare selected.

mRNA-protein fusion system opens the possibility to select among 10¹³different meganuclease variants. This system consists in the creation ofa link between the mRNA and the encoded protein via a puromycin at the3′ end of the mRNA which leads to a covalent mRNA-protein fusion at theend of the translation. Hence, a double-stranded DNA library comprisingthe coding sequence for the meganuclease variants is used regeneratemRNA templates for translation that contain 3′ puromycin. ThemRNA-puromycin conjugates are translated in vitro to generate themRNA-meganuclease fusions. After cDNA synthesis, the fusions are testedfor the ability to bind the immobilized targeted DNA polynucleotide. APCR is then used to generate double-stranded DNA enriched inmeganuclease variants presenting the binding capacity. If the initialmeganuclease is a homodimer, the constant monomer can be introducedeither as DNA or mRNA encoding this monomer or as a monomer protein. Inthis case, an approach with the single chain meganuclease will bepreferably used.

Ribosome display involves a double-stranded DNA library comprising thecoding sequence for the meganuclease variants that is used to generatemRNA templates for translation. After a brief incubation, translation ishalted by addition of Mg²⁺ and incubation al low temperature or additionof translation inhibitor. The ribosome complexes are then tested for theability to bind immobilized targeted DNA polynucleotide. The selectedmRNA is used to construct cDNA and a PCR generates double-stranded DNAenriched in meganuclease variants presenting the binding capacity. Ifthe initial meganuclease is a homodimer, the constant monomer isintroduced either as DNA or mRNA encoding this monomer or as a monomerprotein. In this case, an approach with the single chain meganucleasewill be preferably used.

The targeted DNA sequence can be immobilized on a solid support. Saidsolid support could be a column, paramagnetic beads or a well of amicroplate. For example, the polynucleotides comprising the targeted DNAsequence present a ligand (such as a biotin) at one end, said ligandallowing the immobilization on a solid support bearing the target of theligand (for example, streptavidin if biotin is used).

The selection of the meganuclease variants may usually be monitored by ascreening assay based on the binding or cleavage capacity of thesemeganucleases. However, the selected meganuclease variants can be alsodirectly introduced in a selection step based on the cleavage capacity.

In order to perform the screening assay, the selected meganucleasevariants need to be cloned. If the selection was done with the phagedisplay system, the clone encoding each meganuclease variants can beeasily isolated. If the selection was done by mRNA-protein fusion orribosome display, the selected meganuclease variants have to besubcloned in expression vector.

The screening assays are preferably performed in microplates (96, 384 or1536 wells) in which the targeted DNA polynucleotides are immobilized.After expression of the meganuclease variants, these variants areincubated with the immobilized targeted DNA polynucleotides. Themeganuclease variants expression can be performed either in vivo or invitro, preferably by in vitro expression system. Preferably, themeganuclease variants are purified prior to the incubation with thetargeted polynucleotide. The retained meganuclease variants are thendetected. The detection could be done by several means well known by theman skilled in the art. For example, if phages are used, the detectioncan be done with antibodies against phages (ELISA). Otherwise, theexpression could be done in presence of S35 amino acids in order toobtain radioactive meganucleases. Thus, the binding is estimated by aradio-activity measurement. The invention also considers the othersmeans of detection of DNA binding by meganuclease available to the manskilled in the art.

Optionally, the nucleotide sequences encoding the positively screenedmeganuclease variants are determined, thereby identifying of themeganuclease variants able to bind the targeted DNA sequence.

The positively screened meganuclease variants have to be tested fortheir cleavage capacity. Therefore, said meganuclease variants areincorporated in a cleavage selection and/or screening experiment,preferably an in vivo cleavage screening assay. Optionally, saidmeganuclease variants can be tested by an in vitro cleavage assay.

The screening assay can also be used only for estimate the enrichment inmeganuclease variants presenting the binding capacity. This estimationhelps to decide if a new round of selection based on the bindingcapacity is necessary or if the selected library can be submitted to acleavage selection and/or screening, preferably an in vivo cleavageselection and/or screening.

The selection and screening of meganuclease variants based on thecleavage capacity has to be made in conditions compatible with thecleavage activity. The meganuclease variants used in the selectionand/or screening based on cleavage capacity may be either the initiallibrary of meganuclease variants or the meganuclease variants selectedand/or screened for the binding activity.

If necessary, the selected and/or screened meganuclease variants aresubcloned in an appropriate expression vector for the in vitro and invivo cleavage assay. Such subcloning step can be performed in batch orindividually. More particularly, if the initial meganuclease is a dimer,the subcloning step allows the introduction of the selected library(ies)in a single chain meganuclease structure. If two libraries have beenselected and/or screened for two half recognition and cleavage sites,the subcloning step allows to bring together the two selected librariesin a single chain meganuclease structure.

The general principle of an in vivo selection of the meganucleasevariants based on their cleavage capacity is that the double-strandbreak leads to the activation of a positive selection marker or theinactivation of a negative selection marker.

If the selection is based on the inactivation of a negative selectionmarker, the method involves the use of cell containing an expressionvector comprising the coding sequence for a negative selection markerand the targeted DNA sequence for the desired meganuclease and anexpression vector comprising the library of meganuclease variants.Preferably said expression vector is a plasmid. Preferably said targetedDNA sequence is located either near the negative selection gene or inthe negative selection gene, preferably between the promoter driving theexpression of the negative selection and the ORF. The expression of thenegative selection marker has to be conditional in order to keep thecell alive until the meganuclease variants have the opportunity tocleave. Such a conditional expression can be easily done with aconditional promoter. However, there are other conditional systems thatcould be used. The meganuclease variants are introduced in an expressioncassette. The meganuclease encoding sequence can be operably linked toan inducible promoter or to a constitutive promoter. Of course, thepromoter is compatible with the cell used in the assay. If themeganuclease variant has the capacity to cleave the targeted DNA, thenthe negative selection marker is inactivated, either by deleting thewhole negative marker gene or a part thereof (coding sequence orpromoter) or by degrading the vector. A culture in a negative selectioncondition allows the selection of the cell containing the meganucleasevariants able to cleave the targeted DNA sequence.

The vector comprising the negative selection marker is preferablytransfected before the introduction of the vector encoding themeganuclease variants. Optionally, the vector comprising the negativeselection marker can be conserved in the cell in an episomal form.Alternatively, the vector comprising the negative selection marker andthe vector encoding the meganuclease variants can be cotransfected intothe cell. The cell can be prokaryotic or eukaryotic. Preferably, theprokaryotic cell is E. coli. Preferably, the eukaryotic cell is a yeastcell. The negative selection marker is a protein directly or indirectlytoxic for the cell. For example, the negative selection marker can beselected from the group consisting of toxins, translation inhibitors,barnase, and antibiotic for bacteria, URA3 with 5FOA (5-fluoro-oroticacid) medium and LYS2 with a α-AA medium (alpha-adipic acid) for yeast,and thymidine kinase for superior eukaryotic cells. For an example ofnegative marker selection, see Gruen et al., 2002, Nucleic AcidsResearch, 30, e29; the disclosure of which is incorporated herein byreference.

If the selection is based on the activation of a positive selectionmarker, the method involves the use of cell containing an expressionvector comprising an inactive positive selection marker and the targetedDNA sequence for the desired meganuclease and an expression vectorcomprising the library of meganuclease variants. Optionally, theinactive positive selection marker, the targeted DNA sequence and thelibrary of meganuclease variants can be on the same vector (See WO02/44409). Preferably said expression vector is a plasmid. Themeganuclease variants are introduced in an expression cassette. Themeganuclease encoding sequence can be operably linked to an induciblepromoter or to a constitutive promoter. Of course, the promoter iscompatible with the cell used in the assay. For example, the positiveselection marker can be an antibiotic resistance (e.g. tetracycline,rifampicin and ampicillin resistance) or an auxotrophy marker forbacteria, TRP1, URA3, or an auxotrophy marker for yeast, and neomycineet puromycine for superior eukaryotic cell. Optionally, the positiveselection marker can be an auxotrophy marker compatible with bothbacteria and yeast (e.g. URA3, LYS2, TRP1 and LEU2). The inactivepositive selection marker gene and the targeted DNA sequence have to bearranged so that the double-strand break leads to a rearrangement of themarker in an active positive marker. Two kinds of repair processes canlead to an active positive selection marker, namely single-strandannealing (SSA) or gene conversion (GC).

The in vivo Single-strand annealing recombination test (SSA) is known bythe man skilled in the art and disclosed for example in Rudin et al.(Genetics 1989, 122, 519-534; Fishman-Lobell & Haber (Science 1992, 258,480-4); Lin et al (Mol. Cell. Biol., 1984, 4, 1020-1034) and Rouet et al(Proc. Natl. Acad. Sci. USA, 1994, 91, 6064-6068); the disclosure ofwhich are incorporated herein by reference.

To test the meganuclease variants, an in vivo assay based on SSA in acell, preferably a bacterial or yeast cell has been developed. Forinstance, the method uses a yeast cell. This organism has the advantagethat it recombines naturally its DNA via homologous recombination with ahigh frequency.

This in vivo test is based on the reparation by SSA of a positiveselection marker induced by double-strand break generated by an activemeganuclease variant. The target consists of a modified positiveselection gene with an internal duplication separated by a interveningsequence comprising the targeted DNA sequence. The internal duplicationshould contain at least 50 bp, preferably at least 200 bp. Theefficiency of the SSA test will be increased by the size of the internalduplication. The intervening sequences are at least the targeted DNAsequence. The intervening sequence can optionally comprise a selectionmarker, this marker allowing checking that the cell has not repaired thepositive selection marker by a spontaneous recombination event. Thepositive selection marker gene is preferably operably linked to aconstitutive promoter relating to the cell used in the assay. Accordingto said assay method, the cell will be selected only if a SSA eventoccurs following the double-strand break introduced by an activemeganuclease variant.

Optionally, each vector can comprise a selectable marker to ensure thepresence of the plasmid in the cell. The presence of this selectablemarker is preferable for the assay performed in yeast cell. For example,for yeast, a first construct comprising the target gene can comprise aLeu2 selectable marker allowing transformed yeast to grow on a syntheticmedium that does not contain any Leucine and a second construct cancomprise the Trpl selectable marker allowing transformed yeast to growon a synthetic medium that does not contain any tryptophane.

The vector comprising the positive selection marker is preferablytransfected before the introduction of the vector encoding themeganuclease variants. Optionally, the vector comprising the positiveselection marker can be conserved in the cell in an episomal form.Alternatively, the vector comprising the positive selection marker andthe vector encoding the meganuclease variants can be cotransfected intothe cell.

The in vivo selection of the meganuclease variants can also be performedwith a gene conversion assay. For example, the selection vectorcomprises a first modified positive selection gene with a deletion or amutation and an insertion of the targeted DNA sequence for themeganuclease at the place of the deletion. The positive selection genecan also be inactivated by the interruption of the gene by an insertcomprising the targeted DNA sequence. The selection construct furthercomprises the segment of the positive selection marker gene which hasbeen deleted flanked at each side by the positive selection marker genesequences bordering the deletion. The bordering sequences comprise atleast 100 by of homology with the positive selection marker gene at eachside, preferably at least 300 pb. The double-stand break generated by anactive meganuclease variant in the targeted DNA sequence triggers on agene conversion event resulting in a functional positive selectionmarker gene. This kind of assay is documented in the following articles:Rudin et al (Genetics 1989, 122, 519-534), Fishman-Lobell & Haber(Science 1992, 258, 480-4), Paques & Haber (Mol. Cell. Biol., 1997, 17,6765-6771), the disclosures of which are incorporated herein byreference.

Otherwise, the in vivo selection of the meganuclease variants can beperformed through a recombination assay on chromosomic target. Therecombination can be based on SSA or gene conversion mechanisms.

A first example based on SSA is the following. A modified positiveselection gene with an internal duplication separated by an interveningsequence comprising the targeted DNA sequence for the desiredmeganuclease variant is introduced into the chromosome of the cell. Theinternal duplication should contain at least 50 bp, preferably at least200 bp. The efficiency of the SSA test will be increased by the size ofthe internal duplication. The intervening sequence is at least thetargeted DNA sequence. By transfecting the cell with an expressionconstruct allowing the production of a meganuclease variant in the cell,the repair by homologous recombination of the double-strand breakgenerated by an active meganuclease variant will lead to a functionalpositive selection marker gene.

Another example based on gene conversion is the following. A mutatednon-functional positive selection marker gene comprising the targetedDNA sequence for the desired meganuclease variant is introduced into thechromosome of the cell. Said targeted DNA sequence has to be in thevicinity of the mutation, preferably at less than 1 kb from themutation, more preferably at less than 500 bp, 200 bp, or 100 pbsurrounding the mutation. By transfecting the cell with a fragment ofthe functional positive selection marker gene corresponding to themutation area and an expression construct allowing the production of ameganuclease variant in the cell, the repair by homologous recombinationof the double-strand break generated by an active meganuclease variantwill lead to a functional positive selection marker gene. Alternatively,the fragment of the functional positive selection marker allowing therepair can be integrated on the chromosome. This kind of assay isdocumented in the following articles: Rouet et al (Mol. Cell. Biol.,1994, 14, 8096-8106); Choulika et al (Mol. Cell. Biol., 1995, 15,1968-1973); Donoho et at (Mol. Cell. Biol., 1998, 18, 4070-4078); thedisclosures of which are incorporated herein by reference.

The selected clones comprise a meganuclease variant presenting thecapacity to cleave the targeted DNA sequence. It is preferable tovalidate the selection by a screening assay. This screening assay can beperformed in vivo or in vitro, preferably in vivo.

Optionally, the nucleotide sequences encoding the positively screenedmeganuclease variants are determined, thereby identifying themeganuclease variants able to cleave the targeted DNA sequence.

In order to perform the screening assay, the selected meganucleasevariants need to be cloned and the cleavage assay need to be performedindividually for each clone.

The in vivo cleavage assay for the screening is similar to those usedfor the selection step. It can be based on the inactivation of either anegative selection marker or a reporter gene, or on the activation ofeither a positive selection marker or a reporter gene.

By reporter gene is intended any nucleic acid encoding a product easilyassayed, for example β-galactosidase, luciferase, alkaline phosphatase,green fluorescent protein, tyrosinase, DsRed proteins. The reporter geneis preferably operably linked to a constitutive promoter relating to thecell used in the assay (for example CMV promoter).

Cells used for this screening assay can be prokaryotic, preferably E.coli, or eukaryotic, preferably a yeast cell or a mammalian cell. Moreparticularly, it could be interesting to use mammalian cells for avalidation of a positive meganuclease variant by an ex vivo cleavageassay.

The recognition and cleavage of the targeted DNA sequence or a partthereof by the meganuclease variants can be assayed by any method knownby the man skilled in the art.

One way to test the activity of the meganuclease variants is to use anin vitro cleavage assay on a polynucleotide substrate comprising thetargeted DNA sequence or a part thereof. Said polynucleotide substratecould be a synthetic target site corresponding to:

-   -   the whole targeted DNA site;    -   a half targeted DNA site and a half original site; or,    -   a quarter targeted DNA site and three quarters original site.

Said polynucleotide substrate can be linear or circular and comprisespreferably only one cleavage site. The assayed meganuclease variant isincubated with the polynucleotide substrate in appropriate conditions.The resulting polynucleotides are analyzed by any known method, forexample by electrophoresis on agarose or by chromatography. If thepolynucleotide substrate is a linearized plasmid, the meganucleaseactivity is detected by the apparition of two bands (products) and thedisappearance of the initial full-length substrate band. Preferably,said assayed meganuclease variants are digested by proteinase K, forexample, before the analysis of the resulting polynucleotides. Forinstance, the polynucleotide substrate is prepared by the introductionof a polynucleotide comprising the sequence of the target site in aplasmid by TA or restriction enzyme cloning, optionally followed by thelinearization of the plasmid. Preferably, such linearization is not donein the surrounding of the targeted DNA sequence. See Wang et al, 1997,Nucleic Acid Research, 25, 3767-3776; See Examples, Materials & Methods“in vitro activity assays” section) and the characterization papers ofthe initial homing endonucleases.

Alternatively, such in vitro cleavage assay can be performed withpolynucleotide substrates linked to fluorophores, such substratescomprising the targeted DNA sequence. These polynucleotide substratesare immobilized on a solid support. Said solid support is preferably amicroplate (96, 384 or 1536 wells). For example, the polynucleotidescomprising the targeted DNA sequence present a ligand (such as a biotin)at one end, said ligand allowing the immobilization on a solid supportbearing the target of the ligand (for example, streptavidin if biotin isused). The end opposite to the immobilized end is linked to afluorophore. Cleavage leads to loss of fluorescence by release of thefluorochrome from the solid support.

Otherwise, some in vitro cleavage assays can be based on thefluorescence quenching. A fluorophore (for example, FAM or TAMRA) and aquencher (for example, DABCYL) are located on the polynucleotidesubstrate such as the quencher inhibits the fluorescence emission. Thequenching is abolished when the cleavage by the meganuclease variantsoccurs on the polynucleotide substrates. Several examples of thisquenching assays are detailed in Eisenschmidt et al (2002, Journal ofBiotechnology, 96, 185-191) and WO 02/42497, the disclosure of thesedocuments are incorporated herein by reference.

-   -   Targeting DNA

In a first embodiment of the above uses according to the invention atargeting fragment of DNA (or targeting DNA) comprising a sequence whichmodifies the site of interest flanked by sequences sharing homologies toa targeted locus is also introduced, when necessary (see hereafter theChapter Meganuclease delivery) into the body of said vertebrate (humanor non-human). Preferably, homologous sequences of at least 50 bp,preferably more than 100 by and more preferably more than 200 by areused. The sequence which modifies the site of interest can be thecorrect sequence of a gene for repairing a genetic lesion (genetherapy). Alternatively, it can be any other sequence used to alter thechromosomal DNA in some specific way including a sequence used to modifyof a specific sequence, to attenuate or activate an endogenous gene ofinterest, to inactivate or delete an endogenous gene of interest or partthereof, to introduce a mutation into a site of interest or to introducean exogenous gene or part thereof. Such chromosomal DNA alterations areused for genome engineering (animal models, protein production) or forantiviral therapy.

-   -   Meganuclease Delivery

The meganuclease can be used either as a polypeptide or as apolynucleotide construct encoding said polypeptide under the control ofappropriate transcription regulatory elements including a promoter, forexample a tissue specific and/or inducible promoter. Examples ofinducible promoters are: eukaryotic metallothionine promoter which isinduced by increased levels of heavy metals, prokaryotic lacZ promoterwhich is induced in response to isopropyl-β-D-thiogalacto-pyranoside(IPTG) and eukaryotic heat shock promoter which is induced by increasedtemperature. Examples of tissue specific promoters are skeletal musclecreatine kinase, prostate-specific antigen (PSA), α-antitrypsinprotease, human surfactant (SP) A and B proteins, β-casein and acidicwhey protein genes. It is introduced into somatic cells of anindividual, by any convenient mean well-known to those in the art, aloneor in association with either at least an appropriate vehicle or carrierand/or with the targeting DNA.

According to an advantageous embodiment of the uses according to theinvention, the meganuclease (polypeptide) is associated with:

-   -   liposomes, polyethyleneimine (PEI); in such a case said        association is administered and therefore introduced into        somatic cells target.    -   membrane translocating peptides (Bonetta, 2002, The Sientist,        16, 38; Ford et al, Gene Ther, 2001, 8, 1-4; Wadia & Dowdy,        2002, Curr Opin Biotechnol, 13, 52-56); in such a case, there is        a fusion with said peptides.

Meganucleases can also be introduced into somatic tissue(s) from anindividual according to methods generally known in the art which areappropriate for the particular meganuclease and cell type.

According to another advantageous embodiment of the uses according tothe invention, the meganuclease (polynucleotide encoding saidmeganuclease) and/or the targeting DNA is inserted in a vector. Vectorscomprising targeting DNA and/or nucleic acid encoding a meganuclease canbe introduced into a cell by a variety of methods (e.g., injection,direct uptake, projectile bombardment, liposomes). Meganucleases can bestably or transiently expressed into cells using expression vectors.Techniques of expression in eukaryotic cells are well known to those inthe art. (See Current Protocols in Human Genetics: Chapter 12 “VectorsFor Gene Therapy” & Chapter 13 “Delivery Systems for Gene Therapy”).Optionally, it may be preferable to incorporate a nuclear localizationsignal into the recombinant protein to be sure that it is expressedwithin the nucleus.

Advantageously, the sequence encoding the meganuclease and the targetingDNA are inserted in the same vector.

A vector which can be used in the present invention includes, but is notlimited to, a viral vector, a plasmid, a RNA vector or a linear orcircular DNA or RNA molecule which may consists of a chromosomal, nonchromosomal, semi-synthetic or synthetic DNA. Preferred vectors arethose capable of autonomous replication (episomal vector) and/orexpression of nucleic acids to which they are linked (expressionvectors). Large numbers of suitable vectors are known to those of skillin the art and commercially available.

Viral vectors include retrovirus, adenovirus, parvovirus (e. g.,adenoassociated viruses), coronavirus, negative strand RNA viruses suchas orthomyxovirus (e. g., influenza virus), rhabdovirus (e. g., rabiesand vesicular stomatitis virus), paramyxovirus (e. g. measles andSendai), positive strand RNA viruses such as picornavirus andalphavirus, and double stranded DNA viruses including adenovirus,herpesvirus (e. g., Herpes Simplex virus types 1 and 2, Epstein-Barrvirus, cytomegalovirus), and poxvirus (e. g., vaccinia, fowlpox andcanarypox). Other viruses include Norwalk virus, togavirus, flavivirus,reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example.Examples of retroviruses include: avian leukosissarcoma, mammalianC-type, B-type viruses, Dtype viruses, HTLV-BLV group, lentivirus,spumavirus (Coffin, J. M., Retroviridae: The viruses and theirreplication, In Fundamental Virology, Third Edition, B. N. Fields, etal., Eds., Lippincott-Raven Publishers, Philadelphia, 1996). Otherexamples include murine leukemia viruses, murine sarcoma viruses, mousemammary tumor virus, bovine leukemia virus, feline leukemia virus,feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus,baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkeyvirus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcomavirus and lentiviruses. Other examples of vectors are described, forexample, in McVey et al., U.S. Pat. No. 5,801,030, the teachings ofwhich are incorporated herein by reference.

Vectors can also comprise selectable markers (for example, neomycinphosphotransferase, histidinol dehydrogenase, dihydrofolate reductase,hygromycin phosphotransferase, herpes simplex virus thymidine kinase,adenosine deaminase, glutamine synthetase, and hypoxanthine-guaninephosphoribosyl transferase for eukaryotic cell culture; TRP1 for S.cerevisiae; tetracycline, rifampicin or ampicillin resistance in E.coli; etc . . . ).

Once in a cell, the meganuclease and if present, the vector comprisingtargeting DNA and/or nucleic acid encoding a meganuclease are importedor translocated by the cell from the cytoplasm to the site of action inthe nucleus.

Meganucleases and vectors which comprise targeting DNA homologous to theregion surrounding the cleavage site and/or nucleic acid encoding acustom-made meganuclease can be introduced into an individual usingroutes of administration generally known in the art. Administration maybe topical or internal, or by any other suitable avenue for introducinga therapeutic agent to a patient. Topical administration may be byapplication to the skin, or to the eyes, ears, or nose. Internaladministration may proceed intradermally, subcutaneously,intramuscularly, intraperitoneally, intraarterially or intravenously, orby any other suitable route. It also may in some cases be advantageousto administer a composition of the invention by oral ingestion, byrespiration, rectally, or vaginally.

The meganucleases and vectors can be administered in a pharmaceuticallyacceptable carrier, such as saline, sterile water, Ringer's solution,and isotonic sodium chloride solution. Typically, for therapeuticapplications, the meganucleases will be combined with a pharmaceuticallyacceptable vehicle appropriate to a planned route of administration. Avariety of pharmaceutically acceptable vehicles are well known, fromwhich those that are effective for delivering meganucleases to a site ofinfection may be selected. The HANDBOOK OF PHARMACEUTICAL EXCIPIENTSpublished by the American Pharmaceutical Association is one useful guideto appropriate vehicles for use in the invention. A composition is saidto be a “pharmaceutically acceptable vehicle” if its administration canbe tolerated by the recipient. Sterile phosphate-buffered saline is oneexample of a pharmaceutically acceptable vehicle that is appropriate forintravenous administration. The mode of administration is preferably atthe location of the targeted cells.

The dosage of meganuclease or vector according to the present inventionadministered to an individual, including frequency of administration,will vary depending upon a variety of factors, including mode and routeof administration: size, age, sex, health, body weight and diet of therecipient; nature and extent of symptoms of the disease or disorderbeing treated; kind of concurrent treatment, frequency of treatment, andthe effect desired. For a brief review of pharmaceutical dosage formsand their use, see PHARMACEUTICAL DOSAGE FORMS AND THEIR USE (1985)(Hans Huber Publishers, Berne, Switzerland).

For purposes of therapy, the meganucleases and a pharmaceuticallyacceptable excipient are administered in a therapeutically effectiveamount. Such a combination is said to be administered in a“therapeutically effective amount” if the amount administered isphysiologically significant. An agent is physiologically significant ifits presence results in a detectable change in the physiology of therecipient. In the present context, an agent is physiologicallysignificant if its presence results: in a decrease in the severity ofone or more symptoms of the targeted disease, in a genome correction ofthe lesion or abnormality, or in inhibition of viral infection.

In one embodiment of the uses according to the present invention, themeganuclease is substantially non-immunogenic, i.e., engender little orno adverse immunological response. A variety of methods for amelioratingor eliminating deleterious immunological reactions of this sort can beused in accordance with the invention. In a preferred embodiment, themeganuclease is substantially free of N-formyl methionine. Another wayto avoid unwanted immunological reactions is to conjugate meganucleasesto polyethylene glycol (“PEG”) or polypropylene glycol (“PPG”)(preferably of 500 to 20,000 daltons average molecular weight (MW)).Conjugation with PEG or PPG, as described by Davis et al., (U.S. Pat.No. 4,179,337) for example, can provide non-immunogenic, physiologicallyactive, water soluble endonuclease conjugates with anti-viral activity.Similar methods also using a polyethylene--polypropylene glycolcopolymer are described in Saifer et al. (U.S. Pat. No. 5,006,333).

-   -   Gene Therapy

The use of meganucleases for gene therapy according to the presentinvention varies depending on the type of genetic disease (monogenicrecessive disease, trinucleotide repeats diseases or diseases caused bydominant and compound heterozygous mutations).

Thus, in one embodiment of the present invention, the meganuclease isused in association with a targeting DNA as defined above, comprising asequence to repair the site of interest, for preventing, improving orcuring a monogenetic recessive disease.

In this case, the use of the meganuclease comprises at least the step of(a) inducing in somatic tissue(s) of the individual a double strandedcleavage at a site of interest comprising at least one recognition andcleavage site of said meganuclease, and (b) introducing into theindividual a targeting DNA, wherein said targeting DNA comprises (1) DNAsharing homologies to the region surrounding the cleavage site and (2)DNA which repairs the site of interest upon recombination between thetargeting DNA and the chromosomal DNA. The targeting DNA is introducedinto the individual under conditions appropriate for introduction of thetargeting DNA into the site of interest.

Monogenetic recessive diseases include with no limitations diseasesaffecting the following genes in the corresponding somatic tissues:

-   -   liver: apolipoprotein deficiencies (apoA-I or apoB genes);        familial hypercholesterolemia (FH; LDL receptor gene); Wilson's        disease (WND gene);

Sickle cell anemia (hemoglobin beta gene (HBB)); alpha-1 antitrypsindeficiency (alpha-1 antitrypsin gene); Hereditary hemochromatosis (HFEgene); Hemophilia A or B (Factor IX or X genes); Aminoacidopathies(tyrosinemia (fumarylacetoacetate hydrolase gene (FAH)), phenylketonurea(phenylalanine hydroxylase gene (PAH)), . . . ).

-   -   Lung: Cystic fibrosis (CFTR: Cystic Fibrosis Transmembrane        Regulator)    -   Muscle: Limb-girdle Muscular Dystrophies (α, β, γ or        δ-sarcoglycan genes)    -   Kidney: Polycystic Kidney Disease (PKHDI)    -   Retina: Retinitis pigmentosa (Rhodopsin gene    -   Central Nervous system (CNS): Tay-Sachs disease; Lese-Nyhan        syndrome (HPRT gene).

In another embodiment of the present invention, the meganuclease is usedalone or in association with at least one appropriate vehicle and/orcarrier for preventing, improving or curing a trinucleotide repeatsdisease.

In this case, the trinucleotide repeats ((CGG)n, (CAG)n, or (GAA)n)flanking the meganuclease recognition and cleavage site are deleted byintrachromosomal homologous recombination. More precisely, the use ofthe meganuclease comprises: inducing in somatic tissue(s) of theindividual a double stranded break at a site of interest comprising atleast one recognition and cleavage site of said meganuclease underconditions appropriate for chromosomal DNA homologous to the regionsurrounding the site of cleavage to be deleted into the site of interestand repair of the site of interest (intrachromosomal homologousrecombination).

Trinucleotide repeats diseases include with no limitations, diseasesaffecting the genes as follows:

-   -   Fragile X syndrome: CGG repeat, FMR1 gene    -   Fragile XE syndrome: CCG repeat, FMR2 gene    -   Friedreich ataxia: GAA repeat, X25 gene    -   Myotonic dystrophy: CAG repeat, DMPK gene    -   Huntington disease: CAG repeat, HD gene    -   Spinocerebellar ataxia: CAG repeat, SCA1,2,3,6,7, and 8 genes    -   Haw river syndrome: GAA repeat, DRPLA gene.

In yet another embodiment of the present invention, the meganuclease isused alone or in association with a targeting DNA as defined aboveand/or with at least one appropriate vehicle and/or carrier forpreventing, improving or curing genetic diseases caused by dominant orcompound heterozygous mutations.

Therefore, there are two cases:

1. Meganuclease used alone: the double-strand break at the site of themutation is employed to obtain correction of a genetic lesion via a geneconversion event in which the homologous chromosomal DNA sequences fromanother copy of the gene donates sequences to the sequences where thedouble-stranded break was induced (interchromosomal homologousrecombination). More precisely, the use of the meganuclease comprises:inducing in somatic tissue(s) of the individual a double stranded breakat a site of interest comprising at least one recognition and cleavagesite of said meganuclease under conditions appropriate for chromosomalDNA homologous to the region surrounding the site of cleavage to beintroduced into the site of interest and repair of the site of interest.

2. Meganuclease used in association with a targeting DNA: the use of themeganuclease comprises at least the step of (a) inducing in somatictissue(s) of the individual a double stranded cleavage at a site ofinterest comprising at least one recognition and cleavage site of saidmeganuclease, and (b) introducing into the individual a targeting DNA,wherein said targeting DNA comprises (1) DNA sharing homologies to theregion surrounding the cleavage site and (2) DNA which repairs the siteof interest upon recombination between the targeting DNA and thechromosomal DNA. The targeting DNA is introduced into the individualunder conditions appropriate for introduction of the targeting DNA intothe site of interest. The sequence encoding the meganuclease and thesequence encoding the targeting DNA may be carried by the same vector.

According to the present invention, in both cases, said double-strandedcleavage is induced, either in toto by administration of saidmeganuclease to an individual, or ex vivo by introduction of saidmeganuclease into somatic cells removed from an individual and returnedinto the individual after modification.

Genetic diseases caused by dominant or compound heterozygous mutationsinclude with no limitations: Huntington disease, familialhypercholesterolaemia, familial hyperlipidaemia, oro-facio-digitalsyndrome type 1, dominant otosclerosis, the Bannayan syndrome,hailey-hailey disease, achondroplasia.

-   -   Antiviral Therapy

According to the present invention meganucleases are used astherapeutics in the treatment of viral diseases caused by viruses orretroviruses that present a DNA intermediate. Indeed, many viruses whichinfect eukaryotic cells possess, during at least one part of their lifecycle, genomes that consist of double stranded DNA which can be cleavedreadily by a meganuclease. This strategy involves identification of

DNA sequences within the viral genome that are viral-specific, i.e.,they are not present within the human genome. Once identified,meganucleases that specifically bind and cleave such sequences with highaffinity and specificity can be designed using the method for preparingcustom-made meganucleases as described in the present invention. Thenthe designed meganucleases are used for the treatment of viralinfection.

Meganucleases or expression vector encoding said meganucleases areintroduced into the individual by any convenient mean. When thecustom-made meganucleases are introduced or expressed into the infectedcells, the virus is inactivated and/or deleted. The meganucleasetreatment has no functional impact on healthy cells. Similarly, suchantiviral therapy based on the use of at least one meganuclease could beused to treat organs of an animal dedicated to xenotransplantation.

Any virus that contains a double stranded DNA stage in its life cyclecan be targeted for deletion or inactivation by creating a meganucleasethat recognizes DNA sequences specific of the viral genome. Theseviruses could be in replicative or latent form. They could stay eitherepisomal or integrated in the host's genome.

The double stranded DNA genome viruses are well appropriate to betreated by using meganuclases as defined in the present invention. Amongthem are found the adenoviruses, the herpesviruses, the hepadnaviruses,the papovaviruses, and the poxviruses. Among the herpesviruses are foundherpes simplex virus (HSV), varicella virus (VZV), Epstein-Barr virus(EBV), cytomegalo virus (CMV), herpes virus 6, 7 and 8. Among thehepadnaviruses are found the human hepatitis B virus (HBV). Among thepapovariruses are found papillomavirus (HPV) (i.e. HPV16 or HPV18) andpolyoma virus. Among the adenoviruses are found adenovirus 11 and 21which are involved in acute hemorrhagic cystitis.

The retroviruses are also well appropriate to be treated by usingmeganucleases according to the present invention. Although they are RNAviruses, they are integrated in the host genome as double-stranded DNAform. Among the retroviruses are found the human immunodeficiency virus(HIV) and the human T lymphoma virus (HTLV) (i.e. HTLV1).

According to an advantageous embodiment of the use according to thepresent invention, said virus is selected from HIV, HBV, HTLV, HPV andHSV.

Several above-mentioned viruses are well-known to be involved incarcinogenesis: EBV in Burkitt's lymphoma, other lymphoproliferativedisease and nasopharyngeal carcinoma; herpes virus 8 in Kaposi sarcoma;HBV in hepatocellular carcinoma; HPV in genital cancer; HTLV-1 in T-cellleukemia.

For episomal viruses, a double-strand break introduced in its genomeleads to the linearisation of the genome and its degradation. Examplesof episomal viruses are HSV-1, EBV, and HPV.

For integrated viruses, a double strand break introduced in or near theintegrated viral sequence leads to partial or complete deletion of theintegrated viral sequence. Examples of integrated viruses are HPV, HTLV,HBV, and HIV. Several mechanisms could be involved in the deletion. Adouble-strand break in a chromosome induces a gene conversion with thehomologous chromosome, therefore leading to viral sequence deletion. Ifdirected repeat sequences are present near the double strand break, thebreak could also be repaired by SSA (single strand annealing) leading topartial or complete viral deletion. If two double-strand breaks areintroduced, then the chromosome could also be repaired by end joiningleading to partial or complete deletion of the virus, depending on thepositions of the double-strand breaks. See Example 5 in U.S. Pat. No.5,948,678, the disclosure of which is incorporated herein by reference.

To ensure that the targeted viral DNA sequences are not present in thehost's 3enome, such DNA target sequences should be at least 15nucleotides in length and preferably at least 18 nucleotides in length.As the homing endonuclease present a recognition sequence spanning to12-40 bp, this condition is fulfilled with the custom-made meganucleasesas defined in the present invention. More particularly, I-Cre I homingendonuclease has a 22 by recognition sequence.

Any DNA sequence of viral genomes can be targeted for cleavage bymeganucleases as defined in the present invention. Preferred targetsites include those sequences that are conserved between strains ofvirus and/or which genes are essential for virus propagation orinfectivity. These positions are preferable for at least two reasons.First, essential parts of viruses are less mutated than others.Secondly, it is preferably to target an essential region of the virus tomaximize the inactivation of the virus.

A good target for the custom-made meganuclease could be the viral originof replication (ori) and/or the viral gene encoding an on bindingprotein. Examples of on binding proteins include the HSV-1 UL9 geneproduct, the VZV gene 51 product, the human herpesvirus 6B CH6R geneproduct, the EBV EBNA-1 gene product and the HPV E1 and E2 geneproducts. Other interesting targets for HPV are the genes E6 and E7 asproducts of which are involved in the initiation and maintenance of theproliferative and malignant phenotype. A preferred target is the highlyconserved 62 nucleotides sequence in the pre-core/core region of HPV(E6, E7). Examples of interesting targets for EBV are the genes EBNA andLMP. It could be interesting to target the gene Tax of HTLV-1 whichappears to mediate the oncogenic effects of the virus. For HBV, aninteresting target could be the X gene as the X protein interacts withelements of the DNA repair system and may increase the mutation rate ofp53. For HIV, a preferred target is within TAT, REV, or TAR genes. Theviral targets are not limited to the above-mentioned examples.Optionally, the target DNA could be located in the viral repeatedsequences such as ITR (Inverted Terminal Repeat) and LTR (Long TerminalRepeat).

Preferably, at least two different targeted sites are used. Indeed, asthe main protection of the viruses is their ability to mutate.Therefore, two targeted sites avoid the virus to escape the treatment byusing the custom-made meganucleases, according to the present invention.Moreover, the successive use of different custom-made meganucleases mayavoid the adverse immunologic response. Said different custom-mademeganuclease can present different initial meganucleases, thereforedifferent immunogenicities.

The effectiveness of a meganuclease to inhibit viral propagation andinfection is preferably assessed by in vitro and in vivo assays ofinfection. Such assays can be carried out first in cell culture toestablish the potential of different meganucleases to cleave a viral DNAin a way that deleteriously affects viral propagation. Preliminarystudies of this type are followed by studies in appropriate animalmodels. Finally, clinical studies will be carried out.

Different viruses require different assay systems, since hosts andculture conditions suitable to different viruses vary greatly. However,such appropriate conditions have been described for culturing manyviruses and these conditions can be used to test the effect of exposingvirus and/or host to meganucleases to determine the ability of theendonuclease to inhibit viral infection. For one discussion of cultureconditions for specific viruses see Chapter 17 in Fields and Knipe,Eds., FIELDS VIROLOGY, 2nd Ed., Raven Press, N.Y. (1990).

A host and/or virus can be exposed at various times during a course ofinfection, under varying conditions, in several amounts, and in avariety of vehicles, to mention just a few relevant parameters that canbe varied, to assess the potential of meganuclease to achieve apotentially therapeutic effect.

In addition, in order to tests ex vivo in cultured cells, potentialtherapeutical meganuclease can be tested in animal models to assessprophylactic, ameliorative, therapeutic and/or curative potential,either alone or in conjunction with other therapeutic agents. In somecases, it will not be possible to culture a virus and it will benecessary to perform all biological assays in animal models. It will bereadily appreciated that different animal models will be appropriate todifferent viruses. Any animal model, however, can be used to assess thetherapeutic potential of a meganuclease.

A potentially effective dose of the assayed meganucleases may beadministered to a suitable population of animals, and the effect of themeganucleases on the course of a viral infection may be assessed bycomparison with an appropriate control. Such methods for assessingpharmacological effect are well known in the art and can readily beadapted to determining the therapeutic profile of the meganucleases.

-   -   Genome Engineering

Genome engineering is the set of methods used to induce a change in thegenetic program of a living cell and/or organism. The meganucleasesobtained by the method of the present invention allows rational sitedirected modifications of cell genomes. The purpose of these techniquesis to rewrite chromosomes precisely where they should be modifiedleaving the rest of the genome intact. Fields of applications of thegenome engineering are multiple: animal models generation (knock-in orknock-out), protein production (engineering of production strains,protein production in plant and animals for protein production inmilks), agricultural biotechnology (addition or removal of a trait,marker excision), modification and study of metabolic pathway.

In a first embodiment of the use according to the present invention, itcomprises at least the following steps: 1) introducing a double-strandbreak at the genomic locus comprising at least one recognition andcleavage site of said meganuclease; 2) providing a targeting DNAconstruct comprising the sequence to be introduced flanked by sequencessharing homologies to the targeted locus. Indeed, shared DNA homologiesare located in regions flanking upstream and downstream the site of thebreak in the targeting DNA construct and the DNA that might beintroduced should be located between the two arms. Said meganuclease canbe provided directly to the cell or through an expression vectorcomprising the polynucleotide sequence encoding said meganuclease andsuitable for its expression in the used cell. This strategy is used tointroduce a DNA sequence at the target site, for example to generateknock-in animal models or cell lines that can be used for drug testingor the production of proteins.

In another embodiment of the use according to the present invention itcomprises at least the following steps: 1) introducing a double-strandbreak at the genomic locus comprising at least one recognition andcleavage site of said meganuclease; 2) maintaining under conditionsappropriate for homologous recombination with the chromosomal DNAhomologous to the region surrounding the cleavage site. This strategy isused to delete a DNA sequence at the target site, for example togenerate knock-out animal models for functional genomic studies or forthe generation of appropriate animal models for drug testing.Additionally, knock-outs can be used for the improvement or optimizationof cell lines including the modification of metabolic pathways or thegeneration of cell lines for drug testing.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further illustrated by the additionaldescription and drawings which follows, which refers to examplesillustrating the use of meganucleases for inducing homologousrecombination in somatic tissues according to the invention. It shouldbe understood however that these examples are given only by way ofillustration of the invention and do not constitute in anyway alimitation thereof

FIG. 1 discloses the amino acid sequence of a single chain I-Cre Imeganuclease and one polynucleotide encoding said single chainmeganuclease. In the protein sequence, the two first N-terminal residuesare methionine and alanine (MA), and the three C-terminal residuesalanine, alanine and aspartic acid (AAD). These sequences allow havingDNA coding sequences comprising the NcoI (CCATGG) and EagI (CGGCCG)restriction sites, which are used for cloning into various vectors.

FIG. 2A discloses a polynucleotide called “Natural” encoding the I-Cre Ihoming endonuclease. FIG. 2B discloses a polynucleotide sequence called“Non homologous” encoding the I-Cre I homing endonuclease. FIG. 2Cdiscloses a polynucleotide sequence called “Template” encoding the I-CreI homing endonuclease comprising the mutation D75NJ. Each I-Cre I homingendonuclease has two additional amino acids (MA) at the N terminal endand three additional amino acids (AAD) at the C-terminal ends. FIG. 2Ddiscloses the polynucleotide sequences of the primers, called UlibIfor,UlibIrev, UlibIIfor, and UlibIIrev, used for the generation of thelibraries UlibI and UlibII.

FIG. 3 is a schematic representation of the polynucleotide sequencecalled “Template” encoding the I-Cre I homing endonuclease comprisingthe mutation D75N. The dark arrows indicate the position of the primersUlibIfor, UlibIrev,

UlibiIfor, and UlibIIrev used to generate the two libraries UlibI andUlibIII. D-Helix refers to the LAGLIDADG helix. N75 refers to themutation D75N.

FIG. 4 is a schematic representation of the strategy for the libraryconstruction. Step 1: pET24C-T is a plasmid comprising a polynucleotide<<Template>>. Two PCR amplifications, PCR ulib1 and ulib2, are done witheither UlibIfor and UlibIIrev, or UlibIIfor and UlibIIIrev. The PCRulibl products are cloned in a phagemid pCes4 NHT. The PCR ulib2products are cloned in a plasmid pET24CT. Step 2: Subcloning a fragmentof Ulib2 vector (pET45C-Ulib2) into the Ulib1 phagemid (pCes4-Ulib1).

FIGS. 5A and 5B: COS cells monolayers were transfected with vectorexpressing I Sce-I (FIG. 5B) or with control plasmid (FIG. 5A).Forty-eight (48) hours after transfection cells were infected withrHSV-1 (30 PFU). Two days later monolayer was fixed and stained (X-Gal).Infected cells appeared in blue.

FIG. 6A: cells monolayer was infected with 30 PFU. HSV-1 growth wasquantified by β-galactosidase activity in cell lysate. FIG. 6B, cellmonolayer was infected with 300 PFU. Cell survival was measured byprotein determination in cell lysate. I-Sce I refers to vectorexpressing I-Sce I; I-Sce I(−) refers to a vector in which ORF of ISce-I was inserted in reverse orientation; negative control refers tocontrol plasmid.

FIG. 7A is a schematic representation of recombinant HSV-1 genomic DNA.Cassette containing CMV promoter driving Lac gene was inserted in themajor LAT transcript. I-Sce I restriction site was cloned betweenpromoter and reporter gene. a and b represent primers used for thesemi-quantitative PCR. COS-7 monolayers were transfected with vectorexpressing I-Sce I or with control plasmids. Fourty eight hours aftertransfection cells were infected with rHSV-1 (30 PFU). DNA was extracted1, 2 or 3 days after infection. PCR was carried out as described in<<experimental procedures>>. Std refers to Internal standard; Lac refersto an amplicon of the rHSV-1 Lac gene. I-Sce I refers to vectorexpressing I-Sce I; I-Sce I(−) refers to a vector in which ORF of I-SceI was inserted in reverse orientation; negative control refers tocontrol plasmid. FIG. 7B, PCR quantification of the viral thymidinekinase (TK) gene. PCR was carried out at 2 DNA concentrations. ISce-Irefers to vector expressing I-Sce I; I-Sce I(−) refers to a vector inwhich ORF of I-Sce I was inserted in reverse orientation; negativecontrol refers to control plasmid.

FIG. 8 illustrates the titration of the virus released in the mediumafter infection of the transfected cells. Every day, medium wascollected and fresh medium was added. Viruses were measured by standardplaque assay. I-Sce I refers to vector expressing I-SceI; I-Sce I(−)refers to a vector in which ORF of I-Sce I was inserted in reverseorientation; negative control refers to control plasmid.

FIG. 9 represents the I-Crel DNA target and five related targets.(C1234=SEQ ID NO: 10; C1221=SEQ ID NO: 11; C4334=SEQ ID NO: 12;H1234=SEQ ID NO: 13; H1221=SEQ ID NO: 14; H4334=SEQ ID NO: 15).Conserved positions are in grey boxes.

FIG. 10 illustrates four binding patterns obtained after screening ofthe Lib2 library with six targets. Positives were identified in a firstscreen and confirmed in a second one during which they were assayedeight times (corresponding to the eight solid bars) on each of thetargets (C1234, C1221, C4334, H1234, H1221 and H4334). Histograms areshown for one clone from each class. Targets are described in FIG. 9.

FIG. 11 illustrates the schematic representation of the target vectors.The CpG depleted LacZ gene (LagoZ) is driven by the human elongationfactor 1 alpha promoter. The LagoZ gene is inactivated by the insertionof I-SceI cleavage site. Flanking repeats are represented by openarrows. The length of the homologous sequences are indicated in bold.

FIG. 12 illustrates the effect of the length of homology on singlestrand annealing (SSA) efficiency. Cells monolayers were transfectedwith equimolar amounts of target plasmid bearing different lengths ofhomologous repeat sequences and vector expressing ISce-I or with controlplasmid. Seventy-two hours after transfection cells were collected andβ-galactosidase activity was quantified in cell lysates. (+)I-SceI,cotransfection with vector expressing I-SceI; (−)I-SceI, cotransfectionwith expression vector where the ORF of I-SceI was inserted in thereverse orientation.

FIG. 13A: Cell monolayers were cotransfected with a vector expressing(+)I-SceI or with a control plasmid (−)I-SceI Seventy-two hours aftertransfection cells were fixed and stained (X-Gal). Cells where generepair took place appeared in dark. FIG. 13B: frequency of I-SceIinduced recombination on 70 and 220 by duplication target vectors. Thefrequency is calculated by the ratio of blue cells/transfected cells.

FIG. 14A: X-Gal staining of liver from mice injected with a mixture ofthe target LagoZ gene (30 μg) and an I-SceI expression vector (10 μg).FIG. 14B: X-Gal staining of liver from mice injected with a mixture ofthe target LagoZ gene (30 μg) and an expression vector where the ORF ofI-SceI was inserted in the reverse orientation (10 μg).

FIG. 15A: X-Gal staining of the liver of hemizygote transgenic mice oftwo independent strains infected with the <<Ad.I-SceI>> adenovirus byIV. Five days post-infection, β-galactosidase activity is detected inmultiple cells of the entire liver of 10¹⁰ infectious units infected a<<58A>> hemizygote. In contrast, no β-galactosidase activity could bedetected by X-Gal staining of the livers of <<Ad.control>>-infectedhemizygote or un-infected <<58A>> littermates (data not shown). FIG. 15BFourteen days post-infection, β-galactosidase activity is detected inmultiple cells of the entire liver of 10⁹ infectious units infectedmouse. FIG. 15C: Fourteen days post-infection, β-galactosidase activityis detected in multiple cells of the entire liver of 10¹⁰ infectiousunits infected mouse. Stronger signal is detected in FIG. 15C comparedto FIG. 15B, probably because of the bigger number of cells that wereinfected with the <<Ad.I-SceI>>. In contrast, no β-galactosidaseactivity could be detected by X-Gal staining of the livers ofun-infected <<361>> littermates (data not shown).

FIG. 16: Fluorescent β-galactosidase assay on liver extract. Twoindependent strains of transgenic mice (58 A and 361) were injected with10⁹ or 10¹⁰ PFU of adenovirus expressing I-SceI (Ad.I-SceI) or controlvirus (Ad.control). Mice were sacrified 5 or 14 days post injection,liver was dissected and protein were extracted. 30 _(I)A of liverprotein extract were incubated at 37° C. in presence of Fluoresceindigalactoside (FDG). Bars represent the standard deviation of the assay(two measure experiments with samples of the same extracts). NI, noninjected mice; Ad.I-SceI, mice injected with adenovirus expressingI-SceI; Ad.control, mice injected with control adenovirus.

FIG. 17 represents the transgene, the DNA repair matrix and thesequences of the primers used in example 7. (E=SEQ ID NO: 16; F=SEQ IDNO: 17; G=SEQ ID NO: 18; H=SEQ ID NO: 19; I=SEQ ID NO: 20; Actin S=SEQID NO: 21; Actin R=SEQ ID NO: 22)

FIG. 18 illustrates the RT-PCR analysis of I-SceI-hApo A-I transgenerepair by I-SceI induced gene conversion in mice. Hydrodynamic tail veininjection of naked DNA (I-SceI expression vector and DNA repair matrix)in 3 to 4 weeks old transgenic mice (line 14A and 21). A: 2 kbp DNArepair matrix (RM) and B: 1.5 kbp RM. I-SceI induced gene conversion onI-SceI-hApo A-I transgene in CHO cells was used as PCR positive control.

EXAMPLES Example 1 Single Chain Meganuclease Derived from Dimeric HomingEndonucleases

Some LAGLIDADG homing endonucleases are active as homodimer. Eachmonomer mainly dimerizes through their dodecapeptide motifs. Asingle-chain meganuclease can be engineered by covalently binding twomonomers modified such as to introduce a covalent link between the twosub-units of this enzyme. Preferably, the covalent link is introduced bycreating a peptide bond between the two monomers. However, otherconvenient covalent links are also contemplated. The single-chainmeganuclease preferably comprises two subunits from the same homingendonuclease such as single-chain I-Cre I and single-chain I-Ceu I. Asingle-chain meganuclease has multiple advantages. For example, asingle-chain meganuclease is easier to manipulate. The single-chainmeganuclease is thermodynamically favored, for example for therecognition of the target sequence, compared to a dimer formation. Thesingle-chain meganuclease allows the control of the oligomerisation.

A single chain version of I-CreI (scI-CreI) was modeled and engineered.scI-Crel cleaves its cognate DNA substrate in vitro and induceshomologous recombination both in yeast and mammalian cells.

-   -   Design of the Single Chain I-CreI Meganuclease

I-CreI from Chlamydomonas reinhardtii is a small LAGLIDADG homingendonuclease that dimerizes into a structure similar to that of largermonomer LAGLIDADG homing endonuclease. To engineer a single chainversion of I-CreI (scI-CreI), two I-CreI copies were fused. Thisrequired placing a linker region between the two domains, and asignificant part of the I-CreI protein had to be removed at the end ofthe domain preceding the linker.

The three-dimensional structure of I-DmoI is comparable to that ofI-CreI, with the exception that I-DmoI comprises a linker region thatleads from one apparent domain to the other. The boundary of that linkerfinely matches related main chain atoms of the I-CreI dimer. In thefirst domain, residues 93 to 95 from the third a-helices of I-CreI andI-DmoI (prior to the linker) are structurally equivalent. At thebeginning of the second LAGLIDADG α-helix (second domain), I-DmoIresidues 104 to 106 correspond to I-CreI residues 7 to 9. In addition,Leu95 and G1u105 from I-DmoI have conserved identities in I-CreI, andI-DmoI residue Arg104 aligns with another basic residue in I-CreI(Lys7). Thus, the single chain I-CreI (scI-CreI), was designed byinserting the I-DmoI linker region from residue 94 to 104 (sequenceMLERIRLFNMR) between a first I-CreI domain (terminated at Pro93) and asecond I-CreI domain (starting at Glu8).

Detailed structural analysis of how the new linker connects the scI-CreIprotein domains (in a modeled structure) revealed no potentialincompatibility. For example, the side chains of nonpolar amino acidstaken from I-DmoI, Met94, Ile98 and Phe109 point inside fitting cavitiesof I-CreI. A single mutation was made (P93A), however, to promoteregularity of the backbone in the α-helix prior to the linker region.(See FIG. 1 for amino acids and polynucleotide sequences).

-   -   Materials and Methods

Protein Expression and Purification

His-tagged proteins were over-expressed in E. coli BL21 (DE3) cellsusing pET-24d (+) vectors (Novagen). Induction with IPTG (1 mM), wasperformed at 25° C. Cells were sonicated in a solution of 25 mM HEPES(pH 8) containing protease inhibitors (Complete EDTA-free tablets,Roche) and 5% (v/v) glycerol. Cell lysates were centrifuged twice (15000 g for 30 min). His-tagged proteins were then affinity-purified,using 5 ml Hi-Trap chelating columns (Amersham) loaded with cobalt.Several fractions were collected during elution with a linear gradientof immidazole (up to 0.25 M immidazole, followed by plateau at 0.5 Mimmidazole and 0.5 M NaCl). Protein-rich fractions (determined bySDS-PAGE) were concentrated with a 10 kDa cut-off centriprep Amiconsystem. The resulting sample was eventually purified by exclusionchromatography on a Superdex75 PG Hi-Load 26-60 column (Amersham).Fractions collected were submitted to SDS-PAGE. Selected proteinfractions concentrated and dialyzed against a solution of 25 mM HEPES(pH 7.5) and 20% (v/v) glycerol.

In Vitro Cleavage Assays

pGEM plasmids with single meganuclease DNA target cut sites were firstlinearized with XmnI. Cleavage assays were performed at 37° C. or 65° C.in 12.5 mM HEPES (pH 8), 2.5% (v/v) glycerol and 10 mM MgCl2. Reactionswere stopped by addition of 0.1 volume of 0.1 M Tris-HCl (pH 7.5), 0.25M EDTA, 5% (w/v) SDS, and 0.5 mg/ml proteinase K and incubation at 37°C. for 20 minutes. Reaction products were examined following separationby electrophoresis in 1% agarose gels.

Yeast Colorimetric Assay.

The yeast transformation method has been adapted from previousprotocols. For staining, a classic qualitative X-Gal Agarose OverlayAssay was used. Each plate was covered with 2.5 ml of 1% agarose in 0.1M Sodium Phosphate buffer, pH 7.0, 0.2% SDS, 12% Dimethyl Formamide(DMF), 14 mM β-mercaptoethanol, 0.4% X-Gal, at 60°. Plates wereincubated at 37° C.

Mammalian Cells Assays

COS cells were transfected with Superfect transfection reagentaccordingly to the supplier (Qiagen) protocol. 72 hours aftertransfection, cells were rinsed twice with PBS1× and incubated in lysisbuffer (Tris-HCl 10 mM pH7.5, NaCl 150 mM, Triton X100 0,1%, BSA 0.1mg/ml, protease inhibitors). Lysate was centrifuged and the supernatantused for protein concentration determination and β-galactosidase liquidassay. Typically, 30 μl of extract were combined with 3 μl Mg 100×buffer (MgCl₂ 100 mM, (β-mercaptoethanol 35%), 33 μl ONPG 8 mg/ml and234 μl sodium phosphate 0.1 M pH7.5. After incubation at 37° C., thereaction was stopped with 500 μl of 1 M Na₂CO₃ and OD was measured at415 nm. The relative β-galactosidase activity is determined as afunction of this OD, normalized by the reaction time, and the totalprotein quantity.

-   -   Results: Single Chain I-CreI Cleaves its DNA Substrate in Vitro        and in Living Cells

A synthetic gene corresponding to the new enzyme was engineered and thescI-CreI protein over-expressed in E. coli. The ability of purifiedscI-CreI to cleave DNA substrates in vitro was tested, using linearizedplasmids bearing a copy of the I-Crel homing site. Similarly to parentI-CreI, the novel enzyme cleaves an I-CreI target site at 37° C.

In order to test the functionality of scI-CreI in vivo, an assay tomonitor meganuclease-induced homologous recombination in yeast andmammalian cells was designed. In yeast, Xenopus oocytes and mammaliancells, DNA cleavage between two direct repeats is known to induce a veryhigh level of homologous recombination between the repeats. Therecombination pathway, often referred to as Single-Strand

Annealing (SSA), removes one repeat unit and all intervening sequences.Thus, a SSA reporter vector, with two truncated, non-functional copiesof the bacterial LacZ gene and an I-CreI cut site within the interveningsequence was constructed in a yeast replicative plasmid. Cleavage of thecut site should result in a unique, functional LacZ copy that can beeasily detected by X-gal staining.

The reporter vector was used to transform yeast cells. A small fractionof cells appeared to express functional LacZ, probably due torecombination events during transformation. Co-transformation withplasmids expressing either I-CreI or scI-CreI, in contrast, resulted inblue staining for all plated cells. Even in non-induced conditions(glucose), the residual level of protein was enough to induce SSA,suggesting that scI-CreI, as much as I-CreI, is highly efficient inyeast cells. Furthermore, SSA induction was truly dependent on cleavageof the target cut site by I-CreI proteins, as vectors devoid of thatsite display no increase in β-galactosidase activity compared tobackground levels.

The SSA assay was modified for tests in mammalian cells. The promoterand termination sequences of the reporter and meganuclease expressionplasmid were changed, and plasmid recombination was evaluated in atransient transfection assay. Similar levels of induced recombination (2to 3-fold increase) were observed with either scI-CreI or I-CreI. As inthe yeast experiment, recombination depends on an I-Crel cut sitebetween the repeats, for no increase of the β-galactosidase was observedin the absence of this site.

Another recombination assay, based on recombination between invertedrepeats, was also used to monitor meganuclease-induced recombination inCOS cells. As direct repeats can recombine by SSA, homologousrecombination between indirect repeats requires a gene conversion event.Similar stimulation of gene conversion (3 to 4-fold) was observed witheither scI-CreI or I-CreI. As expected for a true homologousrecombination event, no enhancement was observed in the absence of anhomologous donor template.

Example 2 Custom-made Meganuclease Derived from I-Cre I HomingEndonuclease for HIV-2 Target

-   -   Construction of a Phage-Displayed Library of I-Cre I Variants

In order to engineer new meganuclease with altered specificities, acombinatorial library was constructed by mutagenesis of the I-Cre Ihoming endonuclease replacing DNA binding residues. Selection andscreening applications then enabled to find those variants that wereable to bind a particular, chosen DNA target. For phage display, asI-Cre I is a homodimer, a phagemid vector was required that encoded twoseparate I-Cre I proteins. Only one of the two I-Cre I copies, which wasfused to the phage coat protein p3, was mutated. The resulting proteinlibrary, in phage display format, comprised thus I-Cre Iwild-type/mutant heterodimers. Eight residues (Q26, K28, N30, Y33, Q38,Q44, R68 and R70) capable together of specific interactions with most ofthe bases in a single hal-site within the DNA target were selected. Ourcombinatorial library was obtained by replacing the eight correspondingcodons with a unique degenerated VVK codon. Eventually, mutants in theprotein library corresponded to independant combinations of any of the12 amino acids encoded by the VVK codon (ADEGHKNPQRST) at eight residuepositions. In consequence, the maximal (theoretical) diversity of theprotein library was 12⁸ or 4.29×10^(8.)

-   -   Construction of the Library

First, residue D75, which is shielded from solvent by R68 and R70, wasmutated to N (Asn) in order to remove the likely energetic strain causedby replacements of those two basic residues in the library. Homodimersof mutant D75N (purified from E. coli cells wherein it wasover-expressed using a pET expression vector) were shown to cleave theI-CreI homing site. A phagemid vector was then engineered that encodeswild-type I-CreI (FIG. 2A and 2B: <<Natural>> or <<Non homologous>>) andthe D75N mutant (FIG. 2C: <<Template>>) fused to the phage coat proteinp3 and phage-displayed wild-type/D75N heterodimers were shown to bindthat target DNA.

Second, two intermediate libraries of moderate size have been built:Libl (residues 26, 28, 30, 33 and 38 mutated; theoretical diversity 12⁵or 2.48×10⁵) and Lib2 (residues 44, 68 and 70 mutated; theoreticaldiversity 12³ or 1,7×10³). DNA fragments carrying combinations of thedesired mutations were obtained by PCR (several reactions in 50 μl),using degenerated primers (FIG. 2D: Uliblfor, Uliblrev, Ulibllfor,Ulibllrev) and as DNA template, the D75N gene. Lib1 and Lib2 wereconstructed by ligation of the corresponding PCR products, digested withspecific restriction enzymes, into the D75N mutant gene, within thephagemid vector and within the pET expression vector, respectively.Digestions of vectors and inserts DNA were conducted in two steps(single enzyme digestions) between which the DNA sample was extracted(phenol: chloroform: isoamylalcohol) and EtOH-precipitated. 10 ofdigested vector DNA were used for ligations, with a 5:1 excess of insertDNA. E. coli TG1 cells were transformed with the resulting vectors byelectroporation. To produce a number of cell clones above thetheoretical diversity of either library, up to 35 electroporations ofthe Lib1 ligation samples and 4 electroporations of the Lib2 ligationsamples were necessary. 4×10⁶ (16 times the maximal diversity) and 6×10⁴(35 times the diversity) clones were thus obtained for Lib1 and Lib2,respectively (these numbers were corrected by the number of clonesobtained using ligations done without inserts).

Finally, Lib1 and Lib2 bacterial clones were scraped from plates and thecorresponding plasmid vectors were extracted and purified. The completelibrary was then obtained by sub-cloning a fragment of the Lib2 vectorinto the Lib 1 phagemid vector (see FIG. 4 for a schematic diagram ofthe library construction). Several rounds of DNA 2-step digestions,dephosphorylation, purification, quantification, ligation andelectroporation were performed. After 4 rounds of 150 electroporationshots (which corresponds to 12 ligations of 1.4 μg vector with 0.4 μginsert), 5.5×10⁷ bacterial clones were obtained (after correction forbackground). Bacteria were scraped and stored as a glycerol stock. Inaddition, an aliquot of this glycerol stock was used to inoculate a 200ml culture and the library vector was extracted and purified from thisculture for storage or potential subcloning.

-   -   Material and Methods

Protein Expression and Purification

His-tagged proteins were over-expressed in E. coli BL21 (DE3) cellsusing pET 24d (+) vectors (Novagen). Induction with IPTG (1 mM), wasperformed at 15° C. over 5 night. Cells were cracked for 1 h at 4° C. ina B-Per solution (Bacterial Protein Extraction Reagent, Pierce, 5 ml for200 ml culture cell), containing protease inhibitors (Complete EDTA-freetablets, Roche) and DNase I (80 units)/nuclease (respectively 80 and 60units, Roche). Alternatively, cells were sonicated in a solution of 25mM HEPES (pH 8) containing protease inhibitors (Complete EDTA-freetablets, Roche) and 5% (v/v) glycerol.

Cell lysates were centrifuged twice (15 000 g for 30 min). His-taggedproteins were then affinity-purified, using 1 ml Hi-Trap chelatingcolumns (Amersham) loaded with cobalt. Several fractions were collectedduring elution with a linear gradient of immidazole (up to 0.25 Mimmidazole, followed by plateau at 0.5 M immidazole and 0.5 M NaCl).Protein-rich fractions (determined by SDS-PAGE) were concentrated with a10 kDa cut-off centriprep Amicon system. The resulting sample waseventually purified by exclusion chromatography on a Superdex75 PGHi-Load 26-60 column (Amersham).

Fractions collected were submitted to SDS-PAGE. Selected proteinfractions concentrated and dialyzed against a solution of 25 mM HEPES(pH 7.5) and 20% (v/v) glycerol.

In Vitro Cleavage Assay

pGEM plasmids with single meganuclease DNA target cut sites were firstlinearized with XmnI. Cleavage assays were performed at 37° C. in 12.5mM HEPES (pH 8), 2.5% (v/v) glycerol and 10 mM MgCl₂. Reactions werestopped by addition of 0.1 volume of 0.1 M Tris-HCl (pH 7.5), 0.25 MEDTA, 5% (w/v) SDS, and 0.5 mg/ml proteinase K and incubation at 37° C.for 20 minutes. Reaction products were examined following separation byelectrophoresis in 1% agarose gels.

Phagemid Construction

Phage Display of I-Cre I/D75N heterodimer was obtained by using aphagemid harboring two different ORFs as a bicistron, under the controlof promoter pLac. The first one yields a soluble protein fused to aN-terminal signal sequence directing the product into the periplasmicspace of E. coli Gene I-Cre I WT was cloned into this ORF usingrestriction enzymes ApaLI and AscI. The D75N domain was cloned into thesecond ORF using Nco I and Eag I restriction enzyme, leading to a fusionwith the phage coat protein p3 via a hexahis tag, a C-Myc tag and anamber stop codon. This final phagemid was called pCes1CreT. In asuppressive strain like TG1 or XL1blue, and after infection by a helperphage (e.g. M13K07), D75N-p3 fusions are incorporated in the phage coatand the soluble I-CreI mononers produced in the same compartment willeither dimerize or interact with the displayed D75N domain, therebyproducing particles displaying I-CreI WT/D75N heterodimer.

Phage Production

A 5 mL culture of 2×TY containing 100 μg/ml of ampicillin and 2% glucosewas inoculated with a 1/100 dilution of an overnight culture of bacteriacontaining phagemid pCeslCreT and agitated at 37° C. At an OD₆₀₀ of 0.5,phage helper M13K07 (Pharmacia) was added at a ratio phage: bacteria of20: 1. After 30 min at 37° C. without agitation, the culture wascentrifuged for 10 min at 4000 rpm and the pellet was resuspended in 25ml of 2×TY containing 100 μg/mL Ampicillin and 25 μg/mL Kanamycin, andagitated overnight at 30° C. Culture were centrifuged and supernatantwere used as such in phage ELISA.

PhageELISA

Microtiter plates were coated for 1 h at 37° C. with 100 μl/well ofbiotinylated

BSA at 2 μg/mL in PBS. After several washes in PBS containing 0.1%Tween20 (PBST), wells were incubated with 100 μl/well of streptavidin at10 μg/mL in PBS and incubated for 1 h at RT. Plates were further washedand incubated with biotinylated PCR fragments harboring the target site,at 250 pM in PBS. After 1 h incubation at RT and washing, plates weresaturated with 200 μl/well of PBS containing 3% powder milk and 25 mMCaCl₂ (PMC). PMC was discarded and plates were filled with 80 μl of PMCand 20 μl/well of culture supernatant containing the phage particles.After 1 h of incubation at RT, plates were extensively washed with PBSTand incubated with 100 μl/well of anti 25 M13-HRP conjugated antibody(Pharmacia) diluted 1/5000 in PMC. Plates were incubated for 1 h at RT,washed and incubated with TMB solution (Sigma). The reaction was blockedwith 50 μl/well of 1M H₂O₄. Plates were read at 450 nm. A signal higherthan 3× the background (irrelevant target) can be considered aspositive.

PCR-based Mutagenesis

Plasmid pET24-T45 containing the gene I-CreI D75N was diluted at 1 ng/μlto be used as template for PCR. Degenerated oligonucleotides encodingthe desired randomizations were used to amplify PCR fragments Lib1 andLib2 in 4×50 μl PCR reactions per inserts. PCR products were pooled,EtOH precipitated and resuspended in 50 μl 10 mM Tris.

DNA Digestions

All enzymes and the corresponding buffers were from NEBiolabs.Digestions of up to 10 μg DNA were realised using up to 100 U of a firstrestriction enzyme, at 37° C., in 150 or 500 μl final reaction volume.After 2 h to 6 h, digested DNA was phenol extracted and EtOHprecipitated. Digestion substrates and products were separated usingagarose gel electrophoresis, the desired product being extracted fromthe gel and purified (Nucleospin Extract, Macherey-Nagel). For PCRinserts, digestions were directly purified on Nucleospin columns. Thesecond digestion was then performed in identical conditions. At the endof this second digestion reaction, 0.1 volume of 10× CAP buffer and 0.5μl of CAP were added to the digested vectors, and the samples werefurther incubated for 30 min at 37° C. (The alkaline phosphatase wasinactivated by incubating the sample 10 min at 70° C., after addition ofEDTA). Eventually, the digested and de-phosphorylated DNA was phenolextracted, EtOH precipitated and resuspended in 30 μl of 10 mM Tris pH8.Final DNA concentrations were estimated by comparison of bandintensities in agarose gels after electrophoresis.

Ligations

Large-scale ligations were done at 16° C. (for 16 h) using 1400 ng ofdigested vector and a 5:1 molar excess of digested in 200 μl reactionvolumes and with 4000 U of T4 DNA ligase (NEBiolabs). After ligation,reaction samples were incubated for 20 min at 65° C. to inactivate theligase. The vector DNA was eventually EtOH precipitated and resuspendedat 25 ng/μl in 10 mM Tris pH8.

Electroporations

40 μl of homemade electrocompetent cells TG1 were mixed with 25 ng ofligated DNA (1 μl) in a 2 mm cuvette. After 1 min on ice, cells werepulsed (2.5 Kv, 25 μF, 200 Ohm) and immediately resuspended in 1 ml of2×TY+2% glucose. Cells were placed at 37° C. for 1 h with agitation, andthen plated on large 2×TY plates containing ampicillin (phagemid vector)or kanamycin (pET vector) and 2% glucose and incubated overnight at 30°C. Aliquots were also diluted in 2×TY and plates on small 2×TYAmpicillin glucose plates to obtain isolated colonies allowing thecalculation of library diversities and characterization of severalclones by restriction analysis.

-   -   Selection and Screening of Meganuclease Binding to a        HIV2-derived DNA Target from a Library of I-Cre I Variant using        Phage Display

The goal of this project was to obtain a meganuclease capable of cuttinga sequence found in the genome of HIV2 (GGAAGAAGCCTTAAGACATTTTGA). Thehoming endonuclease I-Cre I was used as a scaffold to build a library of10⁸ variants by randomizing 8 residues located at the DNA-bindinginterface of one I-Cre I monomer (see previous section). This librarywas enriched for binders by several rounds of selection/amplificationusing biotinylated DNA fragments harboring the HIV2 derived target(H1V6335). The selected targets were subsequently screened for bindingusing a phage ELISA.

Materials and Methods

Phagemid Format

A phagemid based on pCesl (pCLS346) was chosen. This plasmid harboredtwo different ORFs as a bicistron, under the control of promoter pLac.The first one yielded a soluble protein fused to a N-terminal signalsequence directing the product into the periplasmic space of E. coli. Inour case, this first product was a wild-type monomer of I-CreI. Thesecond ORF encoded an I-CreI monomer that was fused to the phage coatprotein p3 via a hexahis tag, a C-Myc tag and an amber stop codon. In asuppressive strain like TG1 or XLlblue, and after infection by a helperphage (e.g. M13K07), bacteria harboring this phagemid produces phageparticles and around 1-10% of them displays the recombinant protein ontheir surface.

The monomer fused to p3 and randomized on the DNA-binding interface wasincorporated in the phage coat and the soluble I-CreI monomers producedin the same compartment either dimerize or interact with the displayedmonomer, thereby producing particles displaying I-CreI homodimers (orheterodimers if the monomer fused to p3 was mutated).

Target Production

Two complementary primers encoding the desired sequences but harboringan extra adenosine in 3′ were annealed and ligated into pGEM-t Easy(Promega). After sequencing, a correct clone was chosen as template toPCR amplify a biotinylated 200 pb fragment using the kit KOD (Novagen)and primers SP6 (TTTAGGTGACACTATAGAATAC) and biotT7(biot-TAATACGACTCACTATAGG). The PCR product concentration was estimatedon gel and the fragment was used as such in ELISA or selectionprocedures.

Rescue of the Phagemid Library

A representative aliquot of the library (at least 10× more bacteria thanthe library size) was used to inoculate 50 ml of 2×TY containing 100μg/ml ampicillin and 2% glucose (2TYAG) and the culture was agitated at37° C. At an OD₆₀₀ of 0.5, 5 ml of this culture was infected with helperphage K07 at a ratio phage:bacteria of 20:1 and incubated withoutagitation for 30 min at 37° C. After centrifugation at 4000 rpm for 10min at room temperature (RT), the pellet was resuspended in 25 ml of2×TY containing 100 μg/ml ampicillin and 25 μg/ml kanamycin (2TYAK) andagitated overnight at 30° C. The culture was centrifuged at 4000 rpm for20 min at 4° C. and phage particles were precipitated by the addition of0.2 volume of 20% PEG6000/2.5M NaCl for 1 h on ice.

After centrifugation at 4000 rpm for 20 min at 4° C., the phage pelletwas resuspended in 1 ml of PBS and centrifuged at 10 00 rpm for 5 min.0.2 volume of 20% PEG6000/2.5 M NaCl was added to the supernatant andthe mix was centrifuged at 10 000 rpm to pellet the phage particles.Particles were finally resuspended in 250 μl PBS.

Selection Procedure

Phage particles were diluted in 1 ml of PBS containing 3% dry milk and25 mM CaCl (PMC) and incubated for 1 h at RT. 100 p1 Streptavidin beads(Dynal, 200 μl for the first round) were washed 3× in PMC and blockedfor 1 h in the same buffer.

The biotinylated targets were added to the phage at the indicatedconcentration and the mix was agitated at RT for 1 h. Beads were addedto the mix and incubated at RT for 15 min. Beads were collected on thevial wall using a magnet and washed 10× in PMC containing 0.1% tween.After a final wash in PBS, beads were resuspended in 0.5 ml of 100 mMTriethanolamine pH 12 and incubated for exactly 10 min. The supernatantwere collected and immediately neutralized by 0.5 ml of 1 M Tris pH8. Analiquot of this eluate was serially diluted for titration and with 4 ml2×TY. 5 ml of exponentially growing TG1 cells were added and the mix wasincubated for 30 min at 37° C. without agitation. Cells were plated onlarge 2TYAG plates and incubated overnight at 30° C. Colonies wereresuspended in 2TYAG, adjusted to an OD₆₀₀ of 100 and kept at −80° C.after addition of 15% glycerol.

Screening by Phage ELISA

Isolated colonies from selection outputs were toothpicked into 100 μl of2TYAG in 96 well plates, and agitated overnight at 37° C. Next day, afresh plate containing 100 μl 2TYAG was isolated using a transferdevice. 50 μl of sterile 60% glycerol was added to the overnight plateand this masterplate was stored at −80° C. The fresh plate was agitatedat 37° C. for 2.5 h, rescued by the addition of 2TYAG containing 2×10⁹pfu of helper phage M13K07, incubated for 30 min at 30° C., spun at 1700rpm for 15 min. Cells pellets were resuspended in 150 μl TYAK andagitated overnight at 30° C. After centrifugation, 20 μl of supernatantwas used as described in the previous section.

-   -   Results

Selections

Phage particles displaying I-Cre I variants were produced by infectingbacteria harboring the phagemid library with helper phage M13KO7. Phageparticles were purified by PEG precipitation and incubated with abiotinylated PCR fragment harboring HIV6335 target. After 1 h ofincubation at room temperature, streptavidin-coated magnetic beads wereadded to the solution to retrieve the biotinylated DMA and bound phages.The beads were extensively washed and the bound phages were eluted by pHshock. Bacteria were infected with the eluted phages and plated on large2×TYplates containing ampicillin and 2% glucose. Serial dilutions of analiquot of the eluted phages were used to infect bacteria to calculatethe number of phage particle and obtain isolated colonies.

The day after, bacteria were scrapped from the large plates and storedas glycerol stocks. An aliquot (representative of the diversity) wasused to produce a new batch of phage particles for a second round ofselection.

The stringency of the selections was increased after each round. Thefirst selection was done using 10 nM of biotinylated target. The secondwas done with 400 pM and the washing steps were extended. The thirdround was done using 250 pM and washed more extensively.

As shown on Table 1, the first and second rounds of selection againstthe HIV2 target lead to an output titer characteristic of backgroundvalues (10⁵ to 10⁶ pfu/ml). However, a significant enrichment wasmeasured on round 3.

TABLE 1 Selection titers. C2H6335: selection done on HIV2 target usingthe library described in the other example. Selection Round Input(pfu/ml) Output (pfu/ml) Enrichment 1 6.4 × 10¹¹ 1.4 × 10⁵ NA 2 4.0 ×10¹² 3.0 × 10⁶  3 3 2.8 × 10¹² 6.9 × 10⁷ 33 NA: non applicable.Enrichment is defined as (output n + 1/input n + 1)/(output n/input n).

Screening by Phage ELISA

80 clones randomly picked from each output (as well as unselectedclones) were used to produce phage particles displaying I-CreI variantsin a monoclonal fashion. Supernatants containing the phage particleswere incubated on biotinylated PCR fragment immobilized on plastic viastreptavidin. Bound phages were stained with an HRP-labeled anti p8(major coat protein) monoclonal antibody (Pharmacia). As shown on Table2, no binders were detected among the unselected clones or from theoutputs of the first round of selection. However 60% of clones pickedafter round 2 against are positive against H6335 but negative on anirrelevant target (P1234, target of homing endonuclease PI-SceI). Thisresult is in good agreement with the output titer. Indeed this selectiononly resulted in a mild enrichment, suggesting that a large number ofclones still originate from background. As expected, a third round ofselection lead to 99% of strong binders, which explains the large numberof output phages after this third selection.

TABLE 2 Percentage of positive clones in a ELISA assay directed againstthe I-CreI target (C1234) or the HIV2 derived target (H6335). 77 cloneswere assayed for each output. % positive % positive Selection roundagainst C 1234 against P1234 0 0 0 1 0 0 2 60 0 3 99 0 Round 0:unselected library

Using phage display, new meganucleases were selected from a largelibrary of I-Cre I variants. Selections on biotynilated DNA targets leadto an increase of output titers characteristic of an enrichment formolecules capable of binding the DNA targets. This enrichment wasconfirmed by phage ELISA. These results demonstrate the efficiency ofthe selection and screening methods.

A selection/screen Experiment in Yeast to Identify Novel Meganucleases.

Material and Methods

Bacterial and Yeast Strains

Every subcloning and plasmid preparations are performed in XLI-blue: E.coli provided by Stratagene following standard procedures. Experimentsin S. cerevisiae are done in the following strains:

FYC2-6A: alpha, trp1Δ63, leu2 Δ1, his3 Δ200

FYBL2-7B: a, ura3 Δ851, μrpl Δ63, leu2 Δ1, lys2 Δ202

YASP3 (derived from FYC2-6A): alpha, ura3::SSA-ura3-HIV2-KanR,ade2::SSA-ade2-HIV2-TRP1, μrpl Δ63, leu2Δ, his3 Δ200

Plasmids:

pCLS0279: ADH1 promoter, TRP1 selectable marker and ARS-CEN origin ofreplication, β-galactosidase SSA target, HIV2 6335 cleavage site.

pCLS0569: kanamycin resistance cassette, HIV2 6335, internal fragment ofthe URA3gene.

pCLS0570: kanamycin resistance cassette, HIV2 6335, internal fragment ofthe LYS2 gene.

pCLS0576: TRP1 selectable marker, HIV2 6335, internal fragment of theADE2 gene.

pCLS0047: Galactose inducible promoter, LEU2 selectable marker and 2micron origin of replication.

Results

We decided to perform an in vivo assay in yeast that allows to screenmutagenized I-CreI protein variants with detectable activity towards aspecified target.

A library of mutated I-CreI meganucleases has been first selected by aphage display procedure, resulting in a sub-library enriched forvariants of interest, able to bind the HIV2 6335 target. The insertsfrom this enriched sub-library are subcloned into pCLS0047 under thecontrol of a galactose-inducible promoter, for further selection inyeast. However, we can produce the library directly in the suitableyeast expression vector, and void the phage display step.

We prepared a specific yeast strain (YASP3) containing two reportersystems integrated in chromosomes. These two reporter systems are basedon recombination by Single Strand Annealing (SSA). SSA is induced byspecific cleavage of the HIV2 6335 site.

Namely, we introduced a URA3 SSA target and an ADE2 SSA target. The URA3SSA target was a modified ura3 gene with 2 direct repeats of 600 basepairs separated by 4,3 kb (containing a kanamycin resistance cassetteand the HIV2 6335 cleavage site). The strain was unable to grow on aminimal medium lacking uracile but was resistant to G418. When thistarget was cleaved and recombined properly, the yeast was able to growon media without uracil and was sensitive to G418.

The ADE2 SSA target was a modified ade2 gene with 2 direct repeats of1,1 kb separated by 3,6 kb (containing a tryptophan selectable markerand the HIV2 6335 cleavage site). Because of this mutated ade2 gene, theyeast strain was unable to grow on a minimal medium lacking adenine, butharbored a red color on a medium with a low adenine content. Because ofthe tryptophan selectable marker, it was able to grow on minimal mediawithout tryptophan. When this target was cleaved and recombinedproperly, the yeast was white, able to grow on media without adenine andunable to grow on a minimal medium lacking tryptophan.

Basically, the recipient yeast strain was red (on low adenine medium),G418 resistant, tryptophan prototroph and auxotroph for uracile andadenine. If a specific meganuclease is expressed in this strain andcleaves its target sites, the resulting yeast clone is white, G418sensitive, prototroph for tryptophan and auxotroph for uracile andadenine.

The YASP3 strain was validated by determining the level of spontaneousrecombination of each target alone and of both targets taken together.The URA3 SSA 10 target recombined spontaneously as an uracileprototrophe, G418 sensitive at an approximate 6 10⁻⁴ rate. The ADE2 SSAtarget recombined spontaneously as an adenine prototrophe at anapproximate 2,7 10⁻³ rate. Recombination of both markers occurredspontaneously (resulting in uracile/adenin rototrophes) at anapproximate 10⁻⁶ rate.

A pilot experiment with 1,5×10 in transformants showed no backgroundlevel of uracileladenine prototrophes means that the number of falsepositive clones should be less than 10 after a transformation experimentwith a library that would yield about a million of independent clones.

The library is used to transform YASP3. A classical chemical/heat chockprotocol that routinely gives 10⁶ independent transformants per pg ofDNA was used (Gietz, R. D. and Woods, R. A., 2002) Transformation ofyeast by lithium acetate/single-stranded carrier DNA/polyethylene glycolmethod (Methods Enzymol, 350, 87-96).

Transformation of the strain with the library gives more than 10⁶independent yeast transformants from which a number of clones are ableto grow on a selective medium whithout uracile, leucine and containinggalactose as a carbone source and a low amount of adenine. Among thoseclones, the interesting ones are white indicating that they contain aLEU2 vector allowing the expression of a meganuclease specific for HIV26335 site and that the enzyme is able to cut both URA3 and ADE2reporters.

The positive clones are isolated and screened for their ability toinduce the specific recombination of a plasmidic SSA β-galactosidasetarget (pCLSO279). This plasmidic reporter was a modified LacZ gene with2 direct repeats of 825 base pairs separated by 1,3kb (containing a URA3selectable marker and the HIV2 6335 cleavage site). The vector (whichcan be selected on a medium without tryptophan) is used to transform ayeast strain (FYBL2-7B) and clones are maintained on minimal media 35lacking uracile to maintain the unrecombined LacZ target.

Yeast clones resulting from the selection experiment are mated with theyeast strain containing the SSA β-galactosidase target. Diploids areselected and assayed for induced β-galactosidase activity. A number ofclones are expected to behave as false positives at this step. Theycorrespond to the background level of spontaneous recombination of theURA3 and ADE2 SSA targets. All remaining clones (uracile and adenineauxotrophes able to induce recombination of the SSA-LacZ target) aretrue positives expressing a meganuclease cleaving the HIV2 6335 targetin vivo. Also, other experiments, based on the ones described above, canbe used to determine more precisely the activity of such novel enzymes.

Example Use of Meganuclease for Antiviral Therapy

-   -   Experimental Procedures

Cells

COS-7 cell lines from the american Type culture collection (ATCC) werecultured in DMEM plus 10% fetal bovine serum. PC-12 cells from ATCC weregrown in RPMI1640 supplemented with 10% heat-inactivated horse serum and5% heat-inactivated fetal bovine serum. PC-12 cells were differentiatedas previously described (Su et al., 1999, Journal of Virology,4171-4180). Briefly, cells were seeded on 6 well-plate at 5 10⁴ cellsper well. The following day, cells were incubated in PC-12 mediumcontaining 100 ng/ml of 2,5 S NGF (Invitrogen). Medium was changed everythree days. After 7 days of incubation, undifferentiated cells wereeliminated by adding 2 μM of fluorodeoxyuridine (FdUrd).

Construction of Recombinant HSV-1

HSV-1 was purchased from ATCC. Viruses were propagated on COS-7 cells atlow MOI (0,01PFU/cell). Recombinant virus (rHSV-1) were generated aspreviously described (Lachmann, R.H., Efstathiou, S., 1997, Journal ofVirology, 3197-3207). A 4,6 Kb pstl-bamHl viral genomic DNA fragment wascloned in pUC 19. Based on HSV-1 sequence from data base (ID: NC001806), this region represents nucleotides 118867 to 123460. A cassetteconsisting of a CMV promoter driving Lac gene expression was introducedinto a168 by HpaI deletion. This region is located within the major LATlocus of HSV-1. I-Sce I cleavage site was finally cloned directly afterthe CMV promoter. This construct was used to generate recombinantviruses. Plasmid was linearized by XmnI digestion, and 2 μg of thisplasmid DNA was contransfected with 15 μg of HSV-1 genomic DNA preparedfrom COS-7 infected cells by CaCl₂ method. After 3 or 4 days, infectedcells were harvested and sonicated. Aliquot of the lysed cells were usedto infect COS monolayer. Virus recombinant were selected by overlayingCOS monolayer with 1% agarose in medium containing 300 μg/ml of X-Gal(5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). Blue clones werepicked and further subjected to three round of plaque purification.Presence of the I-Sce I site was confirmed by PCR and in vitro I Sce-Ienzymatic digestion.

Viral Inhibition

6 well-plate were seeded with 2.10⁴ cells per well. The next day COS-7cells were transfected with 0,5 μg is of plasmid expressing ISce-I orcontaining the ISce-I ORF in the opposite orientation by the EFFECTENEmethod according to the manufacturer protocol. We achieved routinely inour laboratory 60 to 70% efficiency using this methodology. Fourthyeight hours later, subconfluent transfected cells were infected withrHSV-1. For infection, rHSV-1 was diluted in PBS containing 1% fetalbovine serum and adsorbed onto cells for 20-40 min at 37°, in humidifiedincubator with 5% CO2. 6 wells-plates were infected at 30 or 300 PFU perwell for respectively viral inhibition or cells survival experiments.Cells were harvested at day 1, 2, and 3 and β-galactosidase activity wasassayed and DNA extracted.

β-galactosidase Activity

Cell monolayer was fixed in 0,5% glutaraldehyde in 100 mM PBS containingmM MgCl₂ at 4° for 10 minutes. After one wash with detergent solution(100 mM PBS, 1 mM MgCl₂, 0,02% Nonidet p-40) cells were incubated at 37°in X-Gal stain solution (10 mM PBS, 1 mM MgCl₂, 150 mM NaCl, 33 mMK₄Fe(CN)₆.3H₂O, 33 mM K₃Fe(CN)₆, 0,1% X-Gal) until color development.Beta-galactosidase activity was also measured on cell extract witho-nitrophenyl-β-D-galactopyrannoside (ONPG) as substrate. Cell monolayerwas washed once with PBS. Cells were then lysed with 10 mM Tris ph 7.5,150 mM NaCl, 1% Triton X-100, protease inhibitors. After 30 minutesincubation on ice cell lysate was centrifuged and β-galactosidase wasassayed. Typically 30 μl of supernatant was combined with 270 μl ofreaction buffer (10 mM PBS; ph 7,5, 1 mM MgCl₂, 0,3% β-mercaptoethanol)containing 800 μg/ml ONPG. The reaction was carried out at 37° andstopped with 0.5 ml of 1 M NaCO₃. Optical density was measured at 415nm. Beta-galactosidase activity is calculated as relative unitnormalized for protein concentration and incubation time.

Semi-quantitative PCR

To measure viral replication of rHSV-1, oligonucleotides were designedto amplify a 217 bp fragment from Lac gene. The standard DNA used inthis assay was generated by cloning this fragment in a Bluescriptplasmid, and by inserting a 50 bp fragment downstream to the 5′oligonucleotide. PCR of the standard produced 267 bp amplicon. Series ofPCR (not shown) were carried out to fix the amount of standard and DNAsample, and the number of cycles to achieve linear response of theamplification. The basic semi-quantitative PCR were carried out in atotal volume of using the READYMIX™ TAQ (Sigma) with 20 pmols of eachprimers and 180 pg of DNA. The tubes were heated for 4 min at 94° andsubjected to 22 cycles: 94° or 1 min, 62° for 50 sec, 72° for 2 min, and72° for 7 min.

Virus Titration

In one series of experiments, the culture medium was collected every dayat days 1, 2,3, and 4, and fresh medium was added. In the other, themedium was not changed during experiment and aliquots were collectedevery day. To monitor for the release of HSV-1 progeny, aliquot ofmedium were titred on COS-7 cells by standard plaque assay.

-   -   Results

The effect of I-Sce I on viral replication was examined using arecombinant Herpes simplex virus carrying a I-Sce I restriction site(rHSV-1). For convenience, rHSV-1 was build with a cassette containingCMV promoter driving the Lac gene. I-Sce I site was inserted at thejunction of the CMV promoter and Lac gene. The expression cassette wascloned by homologous recombination in the major LAT locus which allowedBeta-galactosidase (β-gal) expression during lytic infection in COS-7cell monolayer. Strinkingly transfection of I-Sce I expression vectorbefore viral infection virtually completely inhibited HSV-1 plaqueformation in COS cells (FIG. 5) as shown by X-Gal coloration. Incontrast, control transfection with expression vector containing I-Sce Iopen reading frame in the reverse orientation which did not allow anyfonctional transcript, did not affect viral replication. Furthermore, 48hours after infection, the cells were checked for I-Sce I expression.All the lysis plaques formed in cells monolayer transfected with I-Sce Iexpression vector represented cells which did not expressed I-Sce I (thetransient transfection is about 70% efficient). However, cellsexpressing I-Sce I surrounding the lysis plaque inhibited the viralpropagation. The I-Sce I effect was confirmed by measuring theβ-galactosidase activity in a cell lysate. After infection of COS-7cells monolayer transiently expressing I-Sce I with 30 Pfu per well cellmonolayer was collected at day 1, 2, and 3 post infection and β-gal wasassayed. FIG. 6A shows a drastic decrease of the β-galactosidaseactivity reflecting the inhibition of rHSV-1 replication. The protectiveeffect of I-Sce I over a time course of rHSV-1 infection was evaluatednext. At 3 days after infection, cells transfected with I-Sce Iexpressing vector shown no sign of cytopathic effect whereas controlcultures were completely lysed as shown in FIG. 6B. In an effort toquantify the degree of inhibition of viral DNA replication by I-Sce I,we have set-up a semi-quantitative PCR. Genomic DNA was extracted fromcells at day 1, 2, and 3 after infection. PCR was carried out withprimers a and b (FIG. 7A) generating a 217 by amplicon in Lac gene. Aninternal standard was added in sample before PCR to quantify DNA. Lacgene was virtually not detectable in I-Sce I expressing cells at 3 dayspost-infection (FIG. 7A). In contrast cells that did not received I-SceI expression vector shown high levels of virus DNA. This result wasconfirmed by PCR using primers in viral endogenous gene (FIG. 7B).Amplification of Thymidine Kinase (TK) gene shown that I-Sce I inhibitedthe viral replication. Finally COS-7 cells expressing active I-Sce I orI-Sce I ORF in the reverse orientation were infected with rHSV-1 and theconcentration of virus released in the medium at different time pointswas measured by plaque assay (FIG. 8). Viruses were quantified in arough array at day one when I-Sce I was produced. Viruses production wasstill markly decreased two days after the infection when compared withcells which did not expressed I-Sce I showing that I-Sce I effectivelyinhibited viral replication. This effect was still observed at day threealthough in a lesser extent. Probably the high mutation rate occuringduring viral replication allowed emergence of mutant HSV-1 which wereable to escape the I-Sce I activity.

Taking together, these results demonstrates that I-Sce I and moregenerally meganucleases can be used to inhibit viral infection. The useof custom-made meganuclease or combination of custom-made meganucleasesdesigned to cut specific viral sequences could represent a powerfull newstrategy in the antiviral therapy.

Example 4 Meganuclease with Altered Binding Properties Derived fromI-Crel Homing Endonuclease

The purpose of this experiment was to obtain novel meganucleases bindingtarget sites close to the I-Crel natural target site. A series of 6targets were used (FIG. 9), including the wild-type natural I-Creltarget (named C1234), the HIV2 target described in example 2 (named hereH1234), and four additional targets. These four additional targets are24 by palindromes corresponding to inverted repeats of a 12 by halfI-CreI or HIV2 target site: C1221 and C4334 are inverted repeats of thefirst half and second half, respectively, of the C1234 target; H1221 andH4334 are inverted repeats of the first half and second half,respectively, of the H1234 target.

In contrast with example 2, μhe method used here did not involve anyselection step, but was based on the extensive screening of the Lib2library (see example 2). Three residues (Q44, R68 and R70) capable ofbase specific interactions with the DNA target were selected. Thecombinatorial library was obtained by replacing the three correspondingcodons with a unique degenerated VVK codon. Eventually, mutants in theprotein library corresponded to independant combinations of any of the12 amino acids encoded by the VVK codon (ADEGHKNPQRST) at three residuepositions. In consequence, the maximal (theoretical) diversity of theprotein library was 12³ or 1728.

Materials and Methods

Construction of a Phage-displayed Library of I-Crel Variants.

First, residue D75, which is shielded from solvent by R68 and R70, wasmutated to N (Asn) in order to remove the likely energetic strain causedby replacements of those two basic residues in the library. Homodimersof mutant D75N (purified from E. coli cells wherein it wasover-expressed using a pET expression vector) were shown to cleave theI-Crel homing site. A phagemid vector was then engineered that encodesthe D75N mutant (FIG. 2C: <<Template>>) fused to the phage coat proteinp3 and phage-displayed D75N monomers were shown to bind the I-CreInatural DNA target (C1234 on FIG. 9).

Then, DNA fragments carrying combinations of the desired mutations wereobtained by PCR (several reactions in 50 μl), using degenerated primers(FIG. 2D UlibIIfor, UlibIIrev) and as DNA template, the D75N gene. Lib2was constructed by ligation of the corresponding PCR products, digestedwith specific restriction enzymes, into the D75N mutant gene, within thephagemid vector, as described in example 2.

Screening of Meganucleases Binding to the 6 Different Targets

Screening was performed by Phage ELISA, as described in example 2.

-   -   Results

4560 clones (more than 2.5 μimes the theoretical mutant librarydiversity) were individually picked and screened by phage ELISA with the6 different targets. 28 positives (clones binding one of the sixtargets) were identified. For validation, these 28 clones werere-assayed by phage ELISA, 8 μimes in parallel with the 6 differenttargets; 20 clones were thus confirmed as true positives. Finally, all28 clones were sequenced.

TABLE 3 Sequence of the proteins found in the four different classes.Only amino acids from position 44, 68 and 70 are indicated. Clones foundtwice are labeled with (2). Class I Class II Class III Class IV Q R K(NTQH)N Q R T (2) Unknown Q R R Q R N (RG) (ED) sequence H (KEQ) E Q R AQ Q K (2) Q S R Q N K Q T R (2) Q Q R Q H K D S H Unknown sequence

Four different patterns (ELISA results) could be observed. FIG. 10features one representative example for each one. The first class (ClassI) corresponds to a strong binding of C1234, C1221, C4334 and H4334. Thewild-type protein (QRR) was recovered in this class, showing that ClassI profile is the regular binding profile of the original scaffold. Twovariants were also shown to display such binding (QRK and another yetnot completely identified mutant).

Variants from the second class have lowered their affinity for alltargets, but H4334, since no binding was observed with C1234, C1221 andC4334. Eight different proteins were found to belong to this class, plusa protein which sequence could not be determined. Among the sequencevariants of Class II, five retain the Q44 amino acid from the wild-typesequence, and one of the two arginines in position 68 or 70. However, inone mutant (DSH), none of the amino acids from position 44, 68 and 70has been retained. Class III (4 different proteins) has a more complexpattern, as it retains apparent binding for the C1221 and H4334 target.Finally, one protein (Class IV) retains only a slight binding for targetC 1221 as none of the other targets are bound anymore.

It is difficult to draw conclusions from Class IV, since the residualbinding with C1221 is very low, and sequencing of the unique Class IVmutant has failed. However, comparison of Class II and III with thewild-type profile of Class I clearly shows that the binding specificityhas been altered.

The conclusion is that even from small libraries such as Lib2(complexity 1.7 10³), variants with altered binding profiles can beisolated, as shown in FIG. 10. Therefore, strategies based on screening,starting with larger mutant libraries, should allow the identificationof more dramatic alterations, for instance binding for targets that werenot bound by the initial protein scaffold. In addition, this approachleads to the identification of many different proteins for each profile.An extensive study of this kind should also bring the basis of a betterunderstanding of DNA/meganuclease interactions.

Example 5 Meganuclease-induced Recombination of an ExtrachromosomalReporter in toto using I-Sce I Expressing Plasmid

A-Optimization of the Reporter System

-   -   Experimental Procedures

Vectors Construction

The target vectors are based on a LagoZ expression vector driven bypromoter of the human EF1-alpha gene. This promoter has been shownpreviously to have wide expression spectrum in vivo (Kim, D.W., Uetsuki,T., Kaziro, Y., Yamaguchi, N., Sugano, S, 1990, Gene, 91, 217-223). Thepromoter region includes splice donor and acceptor sites in the 5′untranslated region of the h-EF1-alpha gene-LagoZ is a CpG islanddepleted LacZ gene designed to abolish gene silencing in transgenic mice(Henry I, Forlani S, Vaillant S, Muschler J, Choulika A, Nicolas JF,1999, C R Acad Sci III. 322, 1061-70). To construct target vectors withdifferent lengths of homology, the 3′ fragment of the LagoZ gene wasfirst deleted (about 2000 bp) and replaced by the I-Sce I cleavage site.The 3′ fragments of different lengths were generated by digestion of theparental plasmid. These fragments contained different amounts ofhomology with the 5′ fragment of the LagoZ gene. Finally these DNAfragments were individually cloned adjacent to the I-SceI cleavage site,creating different target vectors with 0, 70, 220, 570, and 1200 by ofhomology, respectively.

Cell Culture

COS-7 and CHO-K1 cell lines from the American Type Culture Collection(ATCC) were cultured in DMEM or Ham's F12K medium respectively plus 10%fetal bovine serum. For I-Sce I induced Single Strand annealing (SSA)assays, cells were seeded in 12 well-plates at a 15.10³ cells per wellone day prior transfection. Transient transfection was carried out thefollowing day with 500 ng of DNA using the EFFECTENE transfection kit(Qiagen). Equimolar amounts of target plasmid and I-SceI expressionvector were used. The next day, medium was replaced and cells wereincubated for an other 72 hours.

β-galactosidase Activity

Cell monolayers were fixed in 0,5% glutaraldehyde in 100 mM PBScontaining 1 mM MgCl₂ at 4° for 10 minutes. After one wash withdetergent solution (100 mM PBS, 1 mM MgCl₂, 0,02% Nonidet p40) cellswere incubated at 37° in X-Gal stain solution (10 mM PBS, 1 mM MgCl₂,150 mM NaCl, 33 mM K₄Fe(CN)₆.3H₂O, 33 mM K₃Fe(CN)₆, 0,1% X-Gal) untilcolor development. Beta-galactosidase activity was also measured in cellextracts with o-nitrophenyl-β-D-galactopyrannoside (ONPG) as asubstrate. Cell monolayers were washed once with PBS. Cells were thenlysed with 10 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, proteaseinhibitors. After 30 minutes incubation on ice, cells lysates werecentrifuged and β-galactosidase was assayed. Typically 30 μl ofsupernatant was combined with 270 μl of reaction buffer (10 mM PBS; pH7,5, 1 mM MgCl₂, 0,3% β-mercaptoethanol) containing 800 μg/ml ONPG. Thereaction was carried out at 37° and stopped with 0,5 ml of 1 M NaCO₃.Optical density was measured at 415 nm. Beta-galactosidase activity iscalculated as relative unit normalized for protein concentration andincubation time.

-   -   Results

When a DNA double-strand break (DSB) is introduced between two repeatedsequences, it induces homologous recombination resulting in a deletionof the repeats, together with all the intervening sequences. Therecombination pathway is often referred to as the single-strandannealing (SSA) pathway. A reporter system was designed to monitormeganuclease-induced SSA in animal models in toto. In order to optimizethe reporter system, the correlation between meganuclease-induced SSAefficiency and repeat length was first examined. Different targetvectors carrying a LagoZ gene containing duplications of various sizeswere constructed (FIG. 11). The presence of the duplication and of theI-SceI cleavage site inactivates the gene. The repair of the LagoZ geneby SSA results in the loss of one repeat and of the cleavage site, andin the restoration of a functional LagoZ gene. LagoZ codes for theβ-galactosidase enzyme which can be detected by colorimetry. Transienttransfection with equimolar amounts of target vector and I-SceIexpression vector or expression vector that doesn't express themeganuclease were carried out in CHO or COS-7 cells. The resultsobtained with the different constructs are presented in FIG. 12. I-SceIinduced DSBs clearly stimulate the SSA repair mechanism. Furthermore,homology of 70 by was sufficient to achieve nearly maximum efficienciesof induced SSA, while the level of spontaneous recombination (withoutI-SceI induced DSB) was minimal. With duplication of 220 by maximumefficiency was achieved while no additional gains in SSA efficiency wereobserved with longer duplications. Similar results were obtained withCOS-7 cells (data not shown). 70 and 220 by of homology gave the bestratio of activity vs background. Because β-galactosidase is assayed incell lysates and one single cell can contain several copies of thetarget plasmid, it is impossible to evaluate the absolute SSA efficiencyby this method. Therefore direct coloration of the cellular monolayerwas performed 72 hours post-transfection (FIG. 13). Virtually no bluecells were detected in the absence of the meganuclease (FIG. 13A). Incontrast, many β-galactosidase-positive cells are present when I-SceI iscotransfected with the target vector, demonstrating the stimulation ofhomologous recombination by meganuclease induced DSB. The efficiency ofI-Sca induced SSA was calculated by counting the blue cells (cells whererecombination has taken place) and comparing it with the number oftransfected cells (cells that effectively received DNA). FIG. 13B showsthat 50 to 60% of the cells undergo homologous recombination when I-SceIis present along with the target vector carrying 70 or 220 byduplications while spontaneous recombination represents less than 0,1%of the events. Thus, the construct with the 70 by and 220 by of homologyas well as the transgene were selected for the animal study.

4B. Meganuclease-induced Recombination of an Extrachromosomal Reporterin toto

-   -   Experimental Procedures

Hydrodynamic-based Transfection in Vivo

Transduction of the mouse liver cells was performed by hydrodynamic tailvein injections as previously described (Zhang, G., Budker, V., Wolff,A., 1999, Human Gene Therapy, 10, 1735-1737; Liu, F.,Song, Y.K., Liu,D., 1999, Gene Therapy, 6, 1258-1266). This method allows efficienttransduction and expression of exogenous genes in animals byadministration of plasmid DNA by tail vein injection. Briefly, DNA ismixed in 1.5 to 2 ml of PBS, which represents 10% of the animal'sweight. Tail vein injections are subsequently performed with a 26-gaugeneedle over a 5-10 sec period using sterile materials and workingconditions. Using such a protocol, almost exclusively liver cells aretransduced, thus the I-SceI-mediated SSA event leading to the correctionof the LagoZ gene was studied in the liver. The I-SceI expressing vectorused is the pCLS 197 corresponding to the I-SceI-coding sequences

(Patent US 5,474,896) under the control of the CMV promoter in a pUCbackbone and is 5737bp long.

OF1 mice weighing fifteen to twenty grams were obtained from CharlesRiver Laboratories, France. A total of twenty micrograms of DNA,containing equal amounts of target vector and either an I-SceIexpression or control vector, was injected into mouse tail veins. Thetarget vector contains the LagoZ gene interrupted by an I-SceI cleavagesite flanked by direct repeat sequences containing 70 by of homology.Control mice were injected with a mixture of the target vector and aplasmid that does not express I-SceI.

β-galactosidase Activity

Three days after injection, mice were euthanized by cervical dislocationand X-Gal stainings of their livers were performed. Livers weredissected out of the animals in cold 1× PBS and the lobes were cut inpieces of about one fourth a centimeter in order to allow a betteraccess of the X-Gal in the tissue. Then liver pieces were placed infresh cold PBS 1× in a 12-well cell culture plate kept on ice, and fixedin 4% paraformaldehyde for 1 hour under agitation at 4° C. Samples werethen washed 3 μimes at room temperature for 30 minutes with wash buffer(100 mM sodium phosphate pH=7.3, 2 mM MgCl₂, 0.01% sodium deoxycholate,0.02% NP-40 by volume). In toto X-Gal staining was performed overnightat 37° C. in staining solution (5 mM potassium ferricyanide, 5 mMpotassium ferrocyanide, lmg/ml X-gal, 20 mM Tris pH=7.3 in wash buffer).Finally samples were washed extensively with PBS and examined undermicroscope. Pictures were taken with a Nikon Coolpix camera under aNikon SMZ 1500 binocular.

-   -   Results

Cellular study has shown that homology of 70 by is sufficient to achievenearly maximal efficiencies of DSB induced SSA, while the level ofspontaneous recombination (without I-SceI induced DSB) is minimal. Thus,in a first attempt to stimulate recombination in vivo, transientexperiments were performed. A mixture of the target vector (30 μg) andeither the I-SceI expression or control plasmid (10 μg) were introducedinto the liver via a hydrodynamic tail vein injection method. FIG. 14shows a magnified picture of liver collected and stained 3 days afterinjection. Blue dots represent cells where a defective LagoZ gene,bearing an I-SceI site flanked by a 70 by duplication, was repaired.After meganuclease induced DSB, the SSA pathway results in the deletionof one repeat and reconstitution of a functional gene. Activeβ-galactosidase encoded by the LagoZ gene can then be detected by X-Galstainings. Furthermore, no gene correction was detected in the absenceof the meganuclease expression vector. These data represent the firstevidence that meganuclease induced recombination can be stimulated inliver and that in toto repair of an extrachromosomal target can beachieved.

Exemple 6 Meganuclease-induced Recombination of a Chromosomal Reporterin toto using I-Sce I Expressing Adenovirus.

In order to demonstrate meganuclease-induced genomic surgery of achromosomal reporter in toto in different mice tissues, the repair ofthe lagoZ gene in toto was tested by transducing cells of several organswith an I-SceI-expressing adenovirus, <<Ad.I-SceI>>. Control transgeniclittermates were infected with a non-I-SceI-expressing adenovirus,<<Ad.control>>. Adenovirus infections in transgenic mice were performedby intraveinous (IV) injections. Repair of the lagoZ gene in toto inseveral tissues was then tested by two methods that detectβ-galactosidase activity in toto: X-gal staining and FDG assays.

-   -   Experimental Procedures

Transgenic Mice

The transgene used for the generation of transgenic founders was aBgIII/NotI fragment of 5919 by carrying the defective LagoZ gene,inactivated by a LagoZ duplication of 70 by or 220 by and the I-SceIcleavage site, under the control of the human elongation factor 1 alphapromoter (See FIG. 11).

Transgenic founder were generated by classical transgenesis, i.e. bymicroinjecting the linear and purified BgIII/NotI fragment describedabove at 500 copies/picolitres into the male pronuclei of fertilized ovaat the one-cell stage derived from the mating of B6D2F1 males andfemales purchased from Elevage Janvier. Microinjections were performedunder a Nikon TE2000 microscope with Normarski DIC with eppendorftransferMan NK2 micromanipulators and eppendorf Femtojet 5247micro-injector. After injections, ova were transferred to surrogatepseudopregnant B6CBAF1/J females (Elevage Janvier) for development anddelivery. Transgenic mice generated by this procedure were identified byPCR and Southern Blot analysis on genomic DNA extracted from tailbiopsies of FO mice. The molecular characterization of the transgeneintegration was done by PCR and Southern Blot analysis.

Then the founder were mated to B6D2F1 mice in order to obtain hemizygotetransgenic F1 animal. Expression of the transgene was tested byperforming an RT-PCR experiment on RNAs extracted from a tail biopsiefrom a transgenic F1 animal using Qiagen RNeasy kit (cat N^(o)74124).Hemizygote F1 mice were then mated to B6D2F1 mice in order to establishan F2 hemizygote transgenic strain.

Two independent strains were used bearing either 220 by or 70 by longlagoZ gene repeated sequences. These transgenic strains are referred asstrain <<361>> and <<58A>>, respectively. The molecular characterizationof the transgene integration showed that the integration is about 5direct repeats of the BgIII/NotI transgene in <<361>> and 2 invertedrepeats plus 5 direct repeats in <<58A>>. Hemizygote mice wereidentified by tail biopsies, genomic DNA extraction and PCR analysis.<<361>> and <<58A>> hemizygote mice were then used for in toto I-SceImediated-lagoZ gene repair and transgenic littermates were used asnegative controls.

Adenovirus-based Transduction in toto

Recombinant type V adenovirus bearing the I-SceI meganuclease codingregion under the control of a CMV promoter, <<Ad.I-SceI>> was providedby Q BIO gene company at 1.58 10¹¹ infectious units concentration scoredby the TCID₅₀ method. The negative adenovirus control <<Ad.control>> wasas well provided by Q BIO gene company at 3.76 10″ infectious unitsconcentration. Recombinant type V adenovirus infections were performedby intraveinous (IV) injections in transgenic mice tail veins.Transgenic mice were weighed and anesthetized before infections byintraperitoneal injection of a mixture of Xylasin (100 mg/kg) andKetamine (10 mg/kg). IV infections were performed with 10¹⁰ infectiousunits/animal in a volume of 400 μl. Infections were performed in 4 to 7weeks-old transgenic mice. Adenovirus-infected mice and uninfectedcontrol littermates were bred in isolator untill sacrificed forβ-galactosidase assays.

β-galactosidase Activity

Adenovirus-infected mice were sacrificed by CO₂ inhalation from 5 to 14days-post-infections (dpi) and their organs were processed forβ-galactosidase assays. About 10% of the liver (8 mm³) was employed forprotein extraction and the remaining 90% was used for β-galactosidase intoto X-gal assays (protocol described previously).

Fluorescent (β-galactosidase assays were incubated at 37° C. in 96 wellplate. The assays were performed in a total volume of 100 μl containing30 μl of protein extract, 1 μM Fluorescein digalactoside (FDG, Sigma),0.1% β-mercaptoethanol, 1 mM MgCl₂, and 100 mM Phosphate buffer, pH 7.0.The plates were scanned on the Fuoroskan Ascent (Labsystem) at 5-minutesintervals. The β-galactosidase activity is calculated as relative unitnormalized for protein concentration and incubation time.

-   -   Results

Two <<58A>> transgenic mice were IV-injected with 10¹⁰ infectious unitsof <<Ad.I-SceI>> adenovirus in order to target a DSB in-between the 70by duplicated lagoZ sequences and induce the repair of the reportergene. At various times post-injection the mice were sacrificed andseveral organs were dissected and analyzed by in toto X-gal assays. Bluestaining was detected as dispersed cells over the entire liver ofinfected mouse euthanized at 5 dpi (FIG. 15A). No staining could bedetected in the other organs tested, i.e. kidneys, spleen, heart andlungs. Two <<58A>> transgenic mouse littermates were used as controls,one IV-injected with 10¹⁰ infectious units of the control adenovirus<<Ad.control>> and the other uninfected. No β-galactosidase activitycould be detected in the liver of either control (data not shown).Similar results were obtained with two <<361>> transgenic mice injectedwith 10⁹ and 10¹⁰ infectious units of the Ad.I-SceI adenovirus (FIG. 15Band 15C respectively). These results were confirmed by measuring theβ-galactosidase activity in liver extract (FIG. 16). A high activity wasdetected in liver of mice injected with Adenovirus expressing I-SceI(Ad.1Sce-I). In contrast, Non-injected mice (NI) shows only a residualbackground activity similar to the activity detected in mice injectedwith the control adenovirus (Ad.control).

The IV-injected mouse with 10¹⁰ infectious units of <<Ad.I-SceI>>adenovirus exhibited more stained liver cells and more β-galactosidaseactivity than the IV-injected mouse with 10⁹ infectious units of<<Ad.I-SceI>> adenovirus. These results suggest that I-SceI-inducedrecombination could be dose dependent and that a better yield of I-SceIinduced recombination could be obtained by increasing theinjected-adenovirus titer. Thus, the detection of I-SceI-induced genomesurgery in other organs reported to be less sensitive to type Vadenovirus infection should be feasible.

Taken together, these data strongly suggest that the reporter generepair was induced by the activity of the I-SceI meganuclease. Thisresult is the first evidence that I-SceI and more generally themeganucleases can be used in toto to induce efficient site-specifichomologous recombination leading to the repair of a chromosomal gene.Thus, these results open applications in the field of gene therapy inmammals.

Exemple 7 Meganuclease-induced Correction of a Mutated Human ApoAI Genein toto

Gene correction would be the safest methodology of gene therapy. Indeedhomologous recombination allows precise modification of chromosomallocus and has been widely used in cell culture. Unfortunately itsefficiency is extremely low and cannot be envisioned, as it is, as atherapeutic tool. However this mechanism has been shown to be enhancedby double strand break (DSB) in the genomic target. So far, invertebrates, the use of this technology has been limited to cellapplications. This is the report for the first time of the use of DSBinduced gene conversion in mammal in toto by direct injection of amixture of meganuclease expression cassette and DNA repair matrix in theblood stream.

The system is based on the repair of a human Apo A-I transgene in micein toto. The apolipoprotein A-I (APO A-I) is the main proteinconstituent of high density lipoprotein (HDL) and plays an importantrole in HDL metabolism. High density lipoproteins have a majorcardio-protective role as the principal mediator of the reversecholesterol transport. The Apo A-I gene is expressed in the liver andthe protein is secreted in the blood. Moreover, Apo A-I deficiency inhuman leads to premature coronary heart disease. All together, thesecriteria make Apo A-I gene a good candidate for the study ofmeganuclease-induced gene correction.

-   -   Experimental Procedures

Transgene

The genomic sequence coding for the human Apo A-I gene was used toconstruct the transgene. Expression of the Apo A-I gene is driven by itsown minimal promotor (328 bp) that has been shown to be sufficient topromote transgene expression in the liver (Walsh et al., J. Biol. Chem.,1989, 264, 6488-6494). Briefly, human Apo A-I gene was obtained by PCRon human liver genomic DNA (Clontech) and cloned in plasmide pUC19. TheI-SceI site, containing two stop codons, was inserted by PCR at thebeginning of exon 4 (FIG. 17). The resulting mutated gene (I-SceI-hApoA-I) encodes a truncated form of the native human APO A-I (80 residuesvs. 267 amino-acids for the wild type APO A-I). All the constructs weresequenced and checked against the human Apo A-I gene sequence.

Generation of Transgenic Mice

The EcoRI/XbaI genomic DNA fragment carrying the mutated human Apo A-Igene was used for the generation of transgenic founders. Microinjectionswere done into fertilized oocytes from breeding of knock out males forthe mouse apo a-I gene (WT KO mice) (The Jackson Laboratory, #002055)and B6SJLF1 females (Janvier). Transgenic founder mice (FO) wereidentified by PCR and Southern blot analysis on genomic DNA extractedfrom tail. FO were then mated to WT KO mice in order to deriveI-SceI-hApo A-I transgenic lines in knock out genetic background for theendogenous murine apo a-I gene. A total of seven independent transgeniclines were studied. The molecular characterization of transgeneintegration was done by Southern blot experiments.

Analysis of transgene expression in each transgenic line was performedby RT-PCR on total RNA extracted from the liver (Trizol Reagent,Invitrogen). In order to avoid cross reaction with the murinetranscript, we used primers specific for the human transgenicI-SceI-hApo A-I cDNA (oligonucleotides E and F, FIG. 17). Actin primerswere used as an internal control.

Hydrodynamic-based Transduction in toto

Transduction of transgenic mouse liver cells in toto was performed byhydrodynamic tail vein injection as previously described (Zhang et al.,1999, Human

Gene Therapy, 10, 1735-1737). 10 to 20 g animals were injected withcircular plasmid DNA in a volume of one tenth their weight in PBS inless than 10 seconds. We used a mixture of 20 or 50 μg of a plasmidcoding for I-SceI under the control of the CMV promoter and the sameamount of a plasmid carrying the DNA repair matrix (2 kbp or 1.5 kbp asdepicted in FIG. 17). We also used 20 or 50 μg of a plasmid carrying theI-SceI expression cassette and the 2 kbp DNA repair matrix in the samevector.

Analysis of Gene Correction

The correction of the transgene in mice after injection of the I-SceIexpression cassette and DNA repair matrix was analyzed by nested PCR ontotal liver RNA reverse transcribed using random hexamers. In order todetect the corrected gene, but not the uncorrected, we used primers setsthat specifically amplified the repaired transgene. The specificity wasachieved by using reverse oligonucleotides spanning the I-SceI site,forward being located outside the repair matrix (oligonucleotides G andH in the first PCR and E and I in the nested one, FIG. 17). Actinprimers were used as an internal control.

-   -   Results

Seven transgenic lines carrying one or several copies of the I-SceI-hapoA-I transgene were used in these experiments. Table 4 summarized themolecular characterization and the expression of the transgene in eachtransgenic line.

TABLE 4 Molecular characterization of transgene integration and level ofexpression in the liver. copy lines integration number expression 14Adirect repeat ~5-10 ++ 14B single 1 + 21 direct repeat ≦5 + 49 directrepeat ≦5 +/− 50 single 1 +/− 66 single 1 + 95 single 1 +

Because transgene expression was very low in lines 49 and 50 furtheranalyses were done on the five other lines. In these experiments, micewere injected with either a mixture of I-SceI-expressing vector and DNArepair matrix or with a vector carrying both I-SceI-expressing cassetteand the DNA repair matrix. The repair of the mutated human Apo A-I genewas monitored by RT-PCR on total liver RNA (FIG. 18) using primersspecifically designed to pair only with the corrected human Apo A-I gene(FIG. 17). As a control, RT-PCR was performed on non-injected transgenicmice or injected with I-SceI expressing vector alone or the DNA repairmatrix alone. No gene correction could be detected with theseexperimental conditions. Furthermore, wild type mice injected witheither a mixture of I-SceI-expressing vector and DNA repair matrix orwith a vector carrying both I-SceI-expressing cassette and the DNArepair matrix did not reveal any PCR-amplified DNA fragment. Incontrast, PCR fragments were specifically visualized in transgenic micewhere I-SceI-expressing cassette and the DNA repair matrix were injected(FIG. 18). The gene correction was detectable in all the transgeniclines tested containing one or several copies of the transgene (Table5).

TABLE 5 RT-PCR analysis of transgene correction after I-SceI expressionvector and repair matrix injections (IV) in mice. Mice where transgenewas repaired/number of injected mice. IV I- I- I- I- RM RM SceI/ SceI/SceI/ SceI/ A B Line RM A RM B RM A Total alone alone alone NI 14A 3/32/2 6/6 11/11 0/2 0/1 0/2 0/6 100%  14B 4/4 0/1 1/3 5/8 0/1 0/3 62% 212/6 1/1 0/1 3/8 0/2 0/2 0/1 0/6 37% 66 2/2 2/2 0/1 100%  95 0/3 1/4 1/70/4 14% Total 22/36 61% WT 0/6 0/1 0/8 0/4 0/1 NI non-injected mice; WT:wild type mice; I-SceI: vector carrying the I-SceI expression cassette;RM A: vector containing the 2 kbp repair matrix; RM B: vector containingthe 1.5 kbp repair matrix and I-SceI-RM A: I-SceI expression cassetteand the 2 kbp repair matrix in the same vector.

Detection of a corrected transgene occurs in 14 to 100% of injected micedepending on the transgenic lines. This heterogeneity probably reflectsthe high variability of the hydrodynamic tail vein injectionmethodology. Simple injection of one plasmid carrying theI-SceI-expressing cassette and the DNA repair matrix or injection of twovectors at the same time gave the same results. Finally, results werealso similar with 20 or 50 μg of DNA injected. These results demonstratethat human Apo A-I gene correction was induced by I-SceI DSB repairmechanism.

These results give evidence that meganuclease-induced gene conversioncan be used to perform in toto genome surgery, and that meganucleasescan be used as drugs for such applications.

1-16. (canceled)
 17. A method of cleaving a targeted double stranded DNAin a liver cell of a mammal in toto, comprising steps of: (a) injectingintravenously a vector comprising a nucleic acid encoding a custom-mademeganuclease specifically recognizing and cleaving a targeted doublestranded DNA, comprising a recognition site 12 to 60 pb in length, and(b) inducing a double strand break in the targeted double stranded DNAby expressing the custom-made meganuclease in the liver cell.
 18. Themethod of claim 17, wherein the vector is injected by hydrodynamicinjection.
 19. The method of claim 17, wherein the double stranded DNAis, or is part of, an infectious agent.
 20. The method of claim 19,wherein the infectious agent is a virus.
 21. The method of claim 20,wherein the virus is selected from the groups consisting of adenoviruse,herpesviruse, hepadnaviruse, papovaviruse and poxviruse.
 22. The methodof claim 20, wherein the virus is in a latent form.
 23. The method ofclaim 20, wherein the virus is in an episomal form.
 24. The method ofclaim 20, wherein the virus is integrated into the mammal's host genome.25. The method of claim 20, wherein the recognition and cleavage site islocated in an ori gene.
 26. The method of claim 21, wherein thehepadnavirus is HBV.
 27. The method of claim 26, wherein the recognitionand cleavage site is located in the gene X of HBV.
 28. The method ofclaim 17, wherein the custom-made meganuclease is a very rare-cuttingendonuclease with a DNA recognition and cleavage site of at least 15 byin length, and preferably at least 18 by in length.
 29. The method ofclaim 17, wherein the custom-made meganuclease is derived from a homingendonuclease.
 30. The method of claim 17, wherein the custom-mademeganuclease is a heterodimer of two fusion proteins.
 31. The method ofclaim 30, wherein the heterodimer comprises a Fokl catalytic domain. 32.The method of claim 31, wherein the custom-made meganuclease comprises azinc finger DNA-binding domain.
 33. The method of claim 17, wherein morethan 1010 units of the vector are injected per mammal.
 34. The method ofclaim 17, wherein the expression of the meganuclease leads to partial orcomplete deletion of the viral sequence.
 35. The method of claim 17,wherein the vector is a recombinant virus.
 36. The method of claim 17,wherein the vector is introduced into the liver cells using liposomes.37. The method of claim 17, wherein the vector is conjugated with PEG,PPG or polyethylene-propylene glycol copolymer.
 38. The method of claim17, wherein the meganuclease is expressed in fusion with a translocatingpeptide or a nuclear localization signal.
 39. The method of claim 17,wherein the mammal is a human.
 40. A therapeutic composition comprisinga meganuclease and a pharmaceutically acceptable carrier or excipientfor intravenous injection.
 41. The therapeutic composition of claim 40,wherein the pharmaceutically acceptable carrier is saline, sterilewater, Ringers solution, or isotonic sodium chloride solution, or amixture of one or more thereof.
 42. The therapeutic composition of claim40, wherein the pharmaceutically acceptable excipient is sterilephosphate buffer saline.
 43. The therapeutic composition of claim 40 forthe treatment of hepatitis or liver cancer.